This makes Ustero the youngest player to ever reach the quarter-finals at just 16 years and one month old, beating Marta Ortega's previous record by three months.
Alonso and Ustero, who are 17 and 16 respectively, upset six-seed Mapi and Majo Sanchez Alayeto with a win in straight sets on Thursday in the round of 16.
They will now look to become only the second qualifiers to ever reach the semi-finals, first done by Fede Chingotto and Juan Tello in 2017 - also in Valladolid.
Ustero and Alonso's win would be the only upset of the day, as the other favourites all managed to win their matches in some capacity.
Matches in chronological order:
Mapi S. Alayeto / Majo S. Alayeto (6) vs Andrea Ustero / Alejandra Alonso
Lucia Sainz / Aranza Osoro (5) vs Lucia Martinez / Carmen Goenaga
Claudia Jensen / Veronica Virseda (8) vs Nuria Vivancos / Carmen Castillon
Virginia Riera / Tamara Icardo (4) vs Eli Amatriain / Sofia Saiz
Jessica Castello / Sofia Araujo (7) vs Teresa Navarro / Marina Lobo
