The addition of antibiotics to intravaginal sponges used to synchronize ewe estrus is currently a recommended practice for the prevention of posttreatment vaginal infections. Application of this antibiotic treatment is usually done without withdrawal periods for milk, but official pharmaceutical instructions do not consider the extra-label use of antibiotics, which could result in the presence of antibiotic residues in milk. To understand the effects of the use of antibiotics on the performance of these hormonal treatments, milk collected from a group of Manchega ewes estrus synchronized by intravaginally inserted progestagen sponges jointly impregnated with antibiotics (benzyl penicillin procaine: 1,000,000 IU/25 sponges plus DH-streptomycin: 1 g/25 sponges) was evaluated for antibiotic residue persistence with 5 types of antibiotic screening tests (BRT, Copan CMT, Delvotest MSC, Eclipse 100, and New SNAP Beta-Lactams). Time to antibiotic residue depletion was established by a logistic regression model, and a significant response to milking order was observed in all methods. Positive or doubtful tests were observed after the insertion of intravaginal sponges for all assay screening tests at the time of the first milking and sometimes afterwards.