The head of the Catalan National Assembly (ANC), Jordi Sánchez, and the head of Òmnium Cultural, Jordi Cuixart, are on trial over sedition charges.
They were jailed without bail on Monday night and could face up to 15 years in prison if convicted.
She said: “The ANC and Òmnium Cultural are the two main pro-independence civil society organisations in Catalonia. These popular organisations have been central to the massive peaceful demonstrations in favour of an independence referendum that we have seen emerge in Catalonia in recent years.
“They were arrested on sedition charges over their alleged role in peaceful protests in the build up to the 1 October independence referendum and on the day of the vote.
“Under Spanish criminal law, a sedition conviction can carry jail sentences of between 4 and 15 years.
“I am deeply concerned that the Spanish High Court has jailed these civil society activists and that they are being prosecuted for organising peaceful demonstrations. Jordi Sánchez and Jordi Cuixart should be released immediately.
“The Spanish State’s response to the Catalan referendum has been very worrying to date.
“The Spanish Government should urgently take up the Catalan Government’s offer of internationally mediated negotiations to find a peaceful resolution to their differences through dialogue,” she added.