"I will return to this house before the end of this month to notify when I have formally triggered Article 50 and begun the process through which the United Kingdom will leave the European Union," she told parliament in an address on Tuesday.
The prime minister told MPs that her timetable for invoking Article 50 of the Lisbon Treaty by the end of March was on track.
"This will be a defining moment for our whole country as we begin to forge the new relationship with Europe and a new role for ourselves in the world," she said.
May's address to parliament on last week's European Council summit came a day after MPs passed the EU Withdrawal Bill, clearing the way for it to receive Royal Assent and become law in the coming days.
The House of Commons voted down amendments by the House of Lords to protect the status of EU nationals and a “meaningful vote” on any Brexit deal. The bill returned to the Lords, who finally backed down after their objections were overturned by MPs.
The speech also came as Scottish First Minister Nicola Sturgeon said she intended to call an independence referendum between the fall of 2018 and the spring of 2019.
Sturgeon said Monday that the UK government had not moved even “an inch towards compromise” with Scotland on Brexit, adding that she would ask permission for a second vote to split from Britain.
In a 2014 referendum, 55 percent of Scottish voters rejected independence from the UK. But Sturgeon said the British government’s decision to withdraw from the EU had brought about a "material change of circumstances."
While Britons overall voted to leave the EU in last year’s Brexit referendum, the Scottish people voted by a margin of 62 percent to 38 percent to remain in the bloc.
In her address, May rejected the notion that Britain would be leaving the EU without Scotland, saying Brexit would "work for the whole of the United Kingdom."
"That's why we have been working closely with the devolved administrations, including the Scottish government - listening to their proposals and recognizing the many areas of common ground, such as protecting workers rights and our security from crime and terrorism,” May said.
"So this is not a moment to play politics and create uncertainty - it's a moment to bring our country together, to honor the will of the British people and shape for them a better Britain," she added.
The prime minister, however, promised to consult with all devolved nations in the Brexit negotiations, a comment that drew laughter from opposition MPs.
Scotland’s Sturgeon has warned there must be "no blocking mechanisms and no Downing Street diktat" to stop her timetable for an independence vote.
Sturgeon hopes her country could retain access to the European single market if it breaks away from the rest of the UK.
May has pursued a divorce from the EU that would also see the UK leaving the bloc’s single market and customs union, arguing that staying in would mean “not leaving at all.”
The single market is a trade agreement between all EU member states which allows free movement of goods, services, capital and people within the bloc.
“I want us to build a new relationship with the EU,” May told the MPs. “A successful and competitive European market in the future will remain in our national interest.”
Labour Leader Jeremy Corbyn accused May’s government of being "complacent," warning the prime minister that “if the wrong decisions are made, we'll pay the price for decades to come."
"Now more than ever we need an inclusive government that listens and acts accordingly," he said. "All the signs are that we have a complacent government."
Corbyn mocked the PM’s claim that "no deal is better than a bad deal," stressing that, "Let me be clear - no deal is a bad deal."
The opposition leader suggested that May’s appeal to stop "uncertainty" was hypocritical because she has indicated that EU citizens might have to leave the UK after Brexit.
"She should tell that to the EU migrants in Britain who have no idea what their future holds because of decisions made by her government," Corbyn said.
"These people are not bargaining chips. They’re mothers, fathers, wives, husbands. They are valued members of our community," he added.
May replied by reiterating her intention to deal with the status of EU nationals at an "early stage" in the negotiations. She emphasized that the future of British citizens in other member states should also be considered.