The 68 year-old had been the mayor of Valencia for 24 years until May last year and was a senator for the conservative Popular Party.
Earlier his year she was named as a suspect in a corruption scandal that alleged she and members of her team were involved in the illegal financing of the party.
A lower court judge initially requested an investigation in April, but Ms Barberá's status as a senator, granting her immunity from the lower courts, meant she could only be investigated by the Supreme Court.
Barberá renounced her membership in the Popular Party in September aftershe was put under investigation.
But said she would stay on in the Senate as an independent, a move whichprotected her from being tried by any tribunal lower than the Supreme Court.
The Supreme Court began investigating Barberá in September and she was called before a judge on Monday to answer allegations.
“I am an honourable person, I am not corrupt, neither economically normorally,” Barberá said in February, adding she felt “boundless pain” because of the suspicions raised about her.
The investigation relates to Operation Taula, which saw 24 people arrested in January and is focused on the activities of local officials in Valencia, Alicante and Castellon.
Paramedics tried to resuscitate her for half an hour after receiving an emergency call from the Villa Real Hotel in Madrid at about 7am.
Politicians payed tribute to a woman who held office in Valencia until May 2015 when her party was ousted by left wing parties.
Prime Minister Mariano Rajoy, who has praised Barberá in the past, said hewas “greatly distressed” by her death.
“She dedicated her life to the Popular Party,” he added as he enteredparliament.
MPs from far-left party Unidos Podemos boycotted a minute of silenceobserved in parliament in honour of Barberá.
“We are not going to take part in a tribute to someone whose path wasmarked by corruption,” the party's leader, Pablo Iglesias, told reporters.