Importance: Antibiotic irrigation of breast implants is widely used internationally, but no clinical study has investigated the pharmacokinetics of antibiotic prophylaxis in the breast implant pocket. Objectives: To evaluate how long locally applied gentamicin, cefazolin, and vancomycin concentrations in the implant pocket remain above the minimum inhibitory concentration (MIC) for the most common bacterial infections and to measure systemic uptake. Design, Setting, and Participants: This prospective cohort study was performed at the Department of Plastic Surgery and Burns Treatment, Rigshospitalet, Copenhagen, Denmark, between October 25, 2021, and September 22, 2022, among 40 patients undergoing implant-based breast reconstruction who were part of the ongoing BREAST-AB trial (Prophylactic Treatment of Breast Implants With a Solution of Gentamicin, Vancomycin and Cefazolin Antibiotics for Women Undergoing Breast Reconstructive Surgery: A Randomized Controlled Trial). Patients were randomized to receive locally applied gentamicin, cefazolin, and vancomycin or placebo. Samples were obtained from the surgical breast drain and blood up to 10 days postoperatively. Exposures: The breast implant and the implant pocket were irrigated with 160 μg/mL of gentamicin, 2000 μg/mL of cefazolin, and 2000 μg/mL of vancomycin in a 200-mL saline solution. Main Outcomes and Measures: The primary outcome was the duration of antibiotic concentrations above the MIC breakpoint for Staphylococcus aureus according to the Clinical and Laboratory Standards Institute: gentamicin, 4 μg/mL; cefazolin, 2 μg/mL; and vancomycin, 2 μg/mL. Secondary outcomes included the time above the MIC for Pseudomonas aeruginosa and other relevant bacteria, as well as systemic uptake. Results: The study included 40 patients (median age, 44.6 years [IQR, 38.3-51.4 years]; median body mass index, 23.9 [IQR, 21.7-25.9]) with a median number of 3 drain samples (range, 1-10 drain samples) and 2 blood samples (range, 0-6 blood samples). Vancomycin and cefazolin remained above the MIC for S aureus significantly longer than gentamicin (gentamicin, 0.9 days [95% CI, 0.5-1.2 days] for blood samples vs 6.9 days [95% CI, 2.9 to 10.9 days] for vancomycin [P =.02] vs 3.7 days [95% CI, 2.2-5.2 days] for cefazolin [P =.002]). The gentamicin level remained above the MIC for P aeruginosa for 1.3 days (95% CI, 1.0-1.5 days). Only cefazolin was detectable in blood samples, albeit in very low concentrations (median concentration, 0.04 μg/mL [range, 0.007-0.1 μg/mL]). Conclusions and Relevance: This study suggests that patients treated with triple-Antibiotic implant irrigation during breast reconstruction receive adequate prophylaxis for S aureus and other common implant-Associated, gram-positive bacteria. However, the protection against P aeruginosa may be inadequate.