Liz Truss has pledged that there will be no second Scottish independence referendum if she wins the Tory leadership contest.
The Foreign Secretary told the Sunday Telegraph that she will refuse another vote if she becomes prime minister.
First Minister Nicola Sturgeon said last month that she intends to hold another independence referendum on October 19 2023.
Earlier this month, Boris Johnson formally rejected a call from the First Minister for Holyrood to be given the power to hold a second vote.
The Prime Minister told Ms Sturgeon in a letter: “I have carefully considered the arguments you set out for a transfer of power from the UK Parliament to the Scottish Parliament to hold another referendum on independence.
“As our country faces unprecedented challenges at home and abroad, I cannot agree that now is the time to return to a question which was clearly answered by the people of Scotland in 2014.”
The Supreme Court is now set to hear arguments on whether a referendum could be held without Westminster’s backing on October 11 and 12.
Ms Truss told the Sunday Telegraph that if succeeds Mr Johnson as prime minister, “there will be no second Scottish independence referendum on my watch”.
“Scottish nationalists accepted that their referendum was a once-in-a-generation opportunity, and I will hold them to that,” she said.
“I will work to strengthen our whole union. As prime minister, I will do what is necessary and right to defend our union, just as I have already done on the Northern Ireland Protocol.”
She added: “The SNP lost the 2014 referendum and Nicola Sturgeon is now leading a campaign of deception to steamroller the UK and break up the Union.
“But I am completely clear that there will be no second Scottish independence referendum on my watch.
“The Scottish Independence Referendum Bill isn’t legal and it will be invalidated if passed by the Scottish Parliament. When Westminster devolved power to Scotland, it did not include the ability to hold valid referenda to break up the Union.
“Any Scotland independence referendum would need to be authorised by the Westminster Parliament. If I become prime minister, I would not grant that authority.”