Ruth Davidson has warned the UK Government not to issue a "blanket refusal" to a second independence referendum if the First Minister calls for one.
Davidson believes such an approach could lead to support for independence growing.
The Scottish Conservatives leader said she looked to the growth in support for Catalonian independence from Spain as an example of what happens when a government refuses outright to hold such a vote.
Madrid has consistently rejected calls for a Catalan referendum and the Spanish constitution forbids the break-up of the union.
Davidson voiced her concerns over such an approach by Downing Street in an interview with STV News.
She said: "In terms of the UK Government's approach [to a second independence referendum], I've said that I don't think there should be a blanket refusal.
"I think you saw in Spain the Spanish government say that to Catalonian independence campaigners and actually support for Catalan independence went through the roof - you had the million man march in Barcelona and stuff.
"I think by even the most generous reckoning the pro-independence marches before the referendum in 2014 hit about 10,000, maybe even 5000 by Police Scotland's figures, so a million is quite a lot more than that and Catalonia is about the same size population wise to Scotland."
Davidson said such a refusal by Downing Street "is something the SNP would like to engineer" to boost support for independence.
She said any attempts by the SNP to call for a second referendum after stating the 2014 vote was a "once in a generation" event are "illegitimate", however.
Davidson said her MSPs will vote against any proposal put forward by the party at Holyrood.
In September, the First Minister said she would bring forward a referendum bill, which is currently being drafted by the Scottish Government, if she concludes "independence is the best or only way to protect Scotland" following the UK's vote to leave the EU.
Nicola Sturgeon said such a referendum was "highly likely" following the EU referendum result.
According to Davidson, such a poll would require a second Edinburgh Agreement between the Scottish and UK governments devolving the power to hold one to Holyrood as well as setting out the question, who can vote in it and the date.
The Edinburgh Central MSP called on Downing Street to be "very clear" about what it would want in such an agreement between Edinburgh and London.
She said: "If there were to be one for whatever reason the SNP has never acknowledged that the need an Edinburgh Agreement.
"I think as a point of law and as well as point of politics they do need one.
"I think the UK Government would be well served to be very clear about what they have to have in it".
In response to Davidson's comments, an SNP spokesman said: "Ruth Davidson is all over the place on this - just as she can't make her mind up whether we should stay in the single market or not.
"The fact is that Scotland voted overwhelmingly to remain in the EU and Theresa May is going out of her way to tell us that Scotland's voice and interests don't matter.
"It's a strange approach from someone who wants to keep the UK together, and Ruth Davidson comes halfway to admitting this in her comments on a second referendum."
Scottish Labour MSP James Kelly reiterated his party's opposition to such a poll being held.
He said: "Scottish Labour couldn't be clearer - we don't want to see a second independence referendum and we will vote against another independence referendum in the lifetime of this Parliament.
Scottish Liberal Democrat leader Willie Rennie said it was"Tory recklessness" which "opened the door for the SNP to talk up a second independence referendum" and that Davidson's comments "will do nothing to calm things down".
Rennie added that the Scottish Conservatives "cannot be trusted" on the union.