Carme Forcadell and five former Catalan lawmakers are accused of sedition, rebellion and misuse of public funds after Catalonia's parliament voted last month to split from Spain.
A judicial source said prosecutors had asked for Forcadell and three others be detained on rebellion charges, which in Spain carries a maximum jail sentence of 30 years.
They are suspected of having followed a "concerted strategy to declare independence", before the official declaration on October 27, deepening Spain's most serious political crisis in decades.
That declaration was annulled Wednesday by Spain's Constitutional Court.
The Catalan crisis has prompted hundreds of businesses to re-register outside the wealthy northeastern region and caused disquiet in a European Union still dealing with Britain's shock decision to leave the bloc.
On Wednesday, a general strike called in Catalonia by a pro-independence union triggered widespread travel chaos, cutting Spain's main highway link to France and the rest of Europe and disrupting trains from Barcelona to Paris, Marseille and Lyon.
Authorities said around 150,000 people were affected.
Encouraged by their charismatic figurehead Carles Puigdemont, Catalan leaders voted to declare unilateral independence from Spain after going ahead with a banned referendum on October 1.
Separatists said 90 percent of voters opted for independence, but critics counter that pro-unity supporters had boycotted a plebiscite that violated Spain's constitution.
In response, the government of Prime Minister Mariano Rajoy suspended Catalanautonomy dismissing its parliament and government, and organised new regional elections for December 21.
A judge in Madrid last week ordered that eight members of Puigdemont's sacked government be detained for their role in the secession drive.
The Supreme Court could also decide to place Forcadell and the former lawmakers in pre-trial detention.
Forcadell's spokesman told reporters that as Catalan parliamentary speaker she didn't "have the freedom to stop a vote" in the chamber on independence.
Outside the court on Thursday a small group of protesters gathered, holding banners against independence.
There were also a handful of separatist supporters, facing opposition cries of "Jail for Puigdemont!"
Puigdemont and four former Catalan ministers are in self-imposed exile in Belgium and are due to appear before a judge next week after Madrid issued a EU-wide warrant for their extradition.
The 54-year-old deposed leader has ignored a summons to appear before a judge in Madrid, saying he wants guarantees he will receive a fair trial.
His presence in the European capital has raised some fears of stoking communal tensions in Belgium after Flemish separatists in the ruling coalition there spoke out in support of Catalan independence.
European Commission chief Jean-Claude Juncker, who was in Spain on Thursday, said that the bloc was "against any form of separatism that weakens Europe".
Despite the crisis, the EU on Thursday upgraded its growth forecast for Spain to a robust 3.1 percent for this year, leading the eurozone's major economies.
EU economics commissioner Pierre Moscovici said that the Catalan independence drive had so far only had a slight impact on Spain's overall economy.
Puigdemont in a letter to the Catalan daily El Punt Avui on Thursday called for "the liberation of political prisoners held by the Spanish state."
Amnesty International however said on Twitter that the jailed ministers could not be considered "political prisoners... as they are accused of acts that may constitute a criminal offence."
Catalonia, a region of 7.5 million people that accounts for a fifth of Spain's GDP, is deeply divided over independence.