Liz Truss has insisted that she will not allow a Scottish independence referendum to be held if she becomes prime minister.
The foreign secretary is competing with former chancellor Rishi Sunak to succeed Boris Johnson in No 10.
And she has pledged that she would not grant any request for a Section 30 Order.
Under current protocol, a Section 30 Order would need to be granted for a referendum to take place.
It would allow for the temporary transfer of the necessary powers from Westminster to Holyrood in order to do so, such as was the case for the 2014 referendum.
The Scottish Government has asked the Supreme Court to rule on whether a vote could be held without the consent of Westminster.
A hearing on the matter will be held in London on October 11 and October 12.
First Minister Nicola Sturgeon has stated her intention to hold a vote on the issue of independence on October 19 next year.
However, Truss has stated that the Scottish Independence Referendum Bill, introduced by the Government at Holyrood, is not legal.
Speaking to the Daily Telegraph, the Conservative leadership candidate made clear her opposition to a future vote.
“Scottish Nationalists accepted that their referendum was a once in a generation opportunity, and I will hold them to that,” said Truss.
“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.”
Truss, who is expected to make a visit to Scotland, outlined her intention to keep the UK together.
“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,” she said.
“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 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.”
Truss added: “As a Conservative and a Unionist, I know that our Union is much more than the sum of its parts.
“If Scotland were to leave the United Kingdom, we would all be worse off – Scottish and English, Welsh and Northern Irish alike.
“I will always stand up for Scotland as a vital part of the UK.
“If I am fortunate enough to be elected, it will be as prime minister for the whole United Kingdom. I intend to keep it that way.”
SNP depute leader Keith Brown described both Truss and Sunak as being “out of touch” with people across Scotland.
“It is crystal clear that the people of Scotland and Scottish democracy mean nothing to either of the contenders for next Tory Prime Minister,” said Brown.
“But people across Scotland will not stand for yet another Tory prime minister we didn’t vote for denying the cast iron mandate to hold an independence referendum of a government and parliament we overwhelmingly voted for.
“With each day that passes, both Tory candidates are showing how out of touch they are with people across Scotland as they prioritise trying to outdo each other on Brexit, ducking their duties to support struggling households and standing in the way of Scotland’s right to choose its future – Scotland deserves so much better.
“Scots voted for the chance to choose a better future in an independence referendum and – whoever becomes the next leader of the Tories – that future as a wealthier, fairer and more equal independent country is more vital than ever.”