Jordi Sanchez of the Catalan National Assembly and Jordi Cuixart of Omnium Cultural are both leaders of their respective groups which played a significant role in encouraging Catalan to claim its independence.
Catalonia’s regional president Carles Puigdemont said: “Sadly, we have political prisoners again.”
Catalan’s foreign minister, Raul Romeva, added: “Leaders of Catalonia’s civil society jailed.
“This is how the new member of UN Human Rights Council responds to our offer of talks.”
A statement from the Madrid court that finalised the decision said there is a risk the Mr Sanchez and Mr Cuixart will “hide, alter or destroy evidence” from rallies organised by each movement in support of Catalan taking back its sovereignty.
The court also claimed that the aim of the rallies was “to stop the Civil Guard” from interfering in the polls.
Spain’s decision to jail the separatist leaders who rallied for independence comes at a time the country is moving closer to imposing central rule over the wealthy region.
Mr Puigdemont missed yesterday’s deadline of clarifying whether Catalonia had declared independence from Spain.
In response, Madrid has given a second ultimatum of Thursday for the region to state its intentions before the possibility of facing direct rules becomes a reality.
Spain’s deputy prime minister, Soraya Sáenz de Santamaría, said Catalonia needs to clarify its position or the government will “take the next steps” in a threat that could see the regions quest for independence halted.
She stated: “No one is denying him the chance for dialogue, but dialogue has to be carried out within the law.
“Puigdemont still has the solution in his hands. He needs to answer yes or no.”
Spain’s Prime Minister, Mariano Rajoy, wrote to Mr Puigdemont yesterday in a further bid to rush the region’s independence decision.
He declared: “Never in all of its history have the citizens of Catalonia enjoyed more liberties or more political and financial autonomy.”
The Catalan government’s campaign to break away from Spain has pushed the country into its worst political crisis since a failed coup attempt in 1981.
Thousands of people have demonstrated in the Catalan capital Barcelona and other Spanish cities both for against independence in recent weeks.
Catalan authorities say voters overwhelmingly backed independence at the referendum, which had been declared illegal by Spain’s Constitutional Court.