Twelve leaders are appearing before the country's Supreme Court in Madrid on charges of rebellion and violating court orders, among others.
The separatist leaders triggered a standoff with the Spanish capital after attempting to push forward with the region's secession in the fall of 2017, sparking the country's worst political crisis since the restoration of democracy in the 1970s.
Catalan separatists have dismissed the trial as a "farce" and said the outcome for the 12 defendants has been pre-determined.
Spanish Supreme Court President Carlos Lesmes has branded the proceedings "the most important trial we've had in democracy."
The defendants -- most of whom are ex-officials of the Catalan regional government -- are being tried for their roles in the botched bid for regional independence.
Despite warnings from the national administration in Madrid that any vote would be unconstitutional, Catalonia went ahead with a referendum, which saw 90% vote in favor of independence, but turnout was low and marred by a violent police crackdown.
The scenes shocked Catalans and reverberated around Europe. Madrid's representative to Catalonia later apologized for the violence.
With the Catalonia question back in the global spotlight, Supreme Court President Lesmes has said he expects proceedings to last around three months.
Perhaps the most prominent name to appear in court is Oriol Junqueras, former deputy leader of Catalonia and head of the separatist Esquerra Republicana de Catalunya (Republican Left of Catalonia) party. Junqueras faces up to 25 years in jail if found guilty.
One notable absence will be that of his erstwhile boss -- and the previous leader of Catalonia -- Carles Puigdemont, who is living in self-imposed exile in Brussels.
Of the 12 defendants, three were released on bail until this week's court proceedings, as they face lesser charges of disobedience and embezzlement and were not considered flight risks.
The Spanish government has been keen to combat any claims of prejudice, publishing a file of documents to illustrate how Spain's justice system is as impartial and fair as its European counterparts.
Included in the documentation were court rankings by the European Commission, the European Court of Human Rights and Transparency International.
A panel of seven Supreme Court justices will preside over the trial and the verdict must be approved by a majority of the panel.
Additionally, the trial will be broadcast live on television so that all can judge the neutrality of the country's highest court.
About 500 witnesses are expected to testify, including Mariano Rajoy, who was the country's Prime Minister during the constitutional crisis.
Upon completion of proceedings, the panel of judges is expected to take some time to reach a verdict. As such, Lesmes has predicted that an outcome is unlikely before late June.
Catalonia is the richest of Spain's 17 autonomous regions. It has its own government and holds considerable powers over healthcare, education and tax collection.
But the region still pays tax to Madrid, and separatists argue that complex mechanisms for redistributing tax revenue are unfair on wealthier areas.
Catalonia has long complained that its revenues subsidize other parts of Spain. The region hosts some 16% of the Spanish population and much of the country's manufacturing and finance sectors.
Catalan nationalists argue that they are a separate nation with their own history, culture and language and that they should have increased fiscal independence.