Among those prosecuted are Catalonia's sacked president Carles Puigdemont and his designated successor, Jordi Turull.
'Junts per Catalonia' member of Catalonia's parliament Jordi Turull during the session on Thursday. Photo: AFP
If found guilty, they face up to 30 years in prison.
Judge Pablo Llarena accused Puigdemont — currently in self-imposed exile in Belgium — of organising the independence referendum in October last year despite a ban from Madrid and “grave risk of violent incidents”.
Turull, a former government spokesman, is already under investigation over Catalonia's secession drive but so far remains free under bail. He was due to appear in court Friday along with several other leading separatists.
But one of them, Marta Rovira, ignored the summons and instead announced she was taking “the road to exile”.
Rovira is deputy leader of the leftwing separatist ERC party, whose chief is currently in jail.
Marta Rovira said she would go into exile. Photo: AFP
She was placed under judicial control in February, but the judge stopped short of putting her behind bars for the duration of an ongoing probe into charges of rebellion, sedition and misuse of public funds.
In a statement released by her party, Rovira said she felt her “freedom of expression was being censored by the courts which intimidate and shamelessly apply political criteria”.
Rovira, who did not specify where she was headed, is the seventh separatist to flee abroad to escape charges.
Separatist parties won regional elections in December called by Madrid after they attempted to secede, retaining their absolute majority in parliament.
But they have still not been able to form a government as their two previous candidates for the presidency proved problematic.
As a result, Catalonia remains under direct rule from Madrid, imposed after the declaration.
The separatist parties failed to elect a new regional president on Thursday after their most radical faction refused to back Turull in a vote of confidence.
A second round parliamentary vote is due to take place on Saturday.