Carme Forcadell and five other deputies appeared in front of Spain's highest tribunal on charges of sedition, rebellion and misuse of public funds – which carry a maximum jail sentence of 30 years -- for aiding the Catalan parliament's secession bid.
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 on Wednesday by Spain's Constitutional Court. Supreme Court judge Pablo Llarena ordered one Catalan lawmaker freed, but the four others apart from Forcadell must pay 25,000 euros within a week to avoid jail.
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 figurehead Carles Puigdemont, Catalan leaders voted to declare unilateral independence from Spain after going ahead with a banned referendum on October 1st.
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 Catalan autonomy dismissing its parliament and government, and organised new regional elections for December 21st.
A judge in Madrid last week ordered that eight members of Puigdemont's sacked government be detained for their role in the secession drive.
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 an 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".
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".
He tweeted that Forcadell had "to sleep in jail for having allowed a democratic debate".