Busquets, 32, captained Spain in Friday's friendly draw with Portugal in Madrid. Spain's players did not train on Sunday but underwent tests, which revealed Busquets' positive result.
The Barcelona midfielder left the national team on Sunday in medical transport as per the protocol while other players, who have yet to be vaccinated, will remain in isolation and have personalised training.
All media activity has been cancelled while Tuesday's international friendly against Lithuania in Madrid will be played by the Spain Under-21 team, whose manager is Luis De la Fuente.
Aside from Busquets, eight players -- Jose Gaya, Mikel Oyarzabal, Thiago, Adama Traore, Aymeric Laporte, Eric Garcia, Fabian Ruiz and Ferran Torres -- in Spain's squad have tested positive for COVID-19 since the start of the pandemic.
UEFA allows for a change of a player from the registered list due to injury or COVID-19 but does not allow for the increase in the number of players called up, which is 24.
On Monday, Spain called up four new players to take part in a parallel training bubble following Busquets's positive COVID-19 test at the weekend, the national team said.
The new additions -- Rodrigo Moreno (Leeds United), Pablo Fornals (West Ham United), Carlos Soler (Valencia) and Brais Mendez (Celta Vigo) -- will not stay at Spain's Las Rozas training base or be involved with the rest of the preparations of the squad, who are currently isolating.
"As a provision to the possible consequences that could come from Sergio Busquets's positive test, the coaching staff have decided to call up a group of four players to complement the training sessions over the next few days under the guidance of [manager] Luis Enrique in a parallel bubble," said a statement.