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Objective: To compare antimicrobial activity as demonstrated by the zone of inhibition (ZOI) produced by antibiotic-impregnated calcium sulfate (CaSO 4 ) beads after storage for 0, 3, 6, 9, and 12 months., Study Design: Controlled laboratory study., Sample Population: Three-millimeter diameter CaSO 4 beads impregnated with vancomycin (125 mg/mL), or amikacin (250 mg/mL), or without antibiotic (control)., Methods: Calcium sulfate beads were created at the onset of the study. Individual beads were separated in sterile containers and stored in a closed cabinet at room temperature and humidity for 0, 3, 6, 9, or 12 months until testing. The ZOI against methicillin-resistant Staphylococcus pseudintermedius, methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa was recorded with serial replating on a fresh lawn of bacteria every 24 h until beads failed to produce a ZOI. The ZOIs and their changes were compared with mixed-effects linear models. Eluted concentrations of vancomycin measured with high-performance liquid chromatography were reported., Results: At 24 h, ZOIs were comparable regardless of time since formulation, except vancomycin against P. aeruginosa, which failed to generate a ZOI. The daily changes of ZOI and duration of activity of antibiotics did not vary between storage length (p > .05). There was no consistent change in eluted drug concentration between storage length of beads., Conclusion: Light protected storage at room temperature for up to 12 months did not impair the in vitro activity of antibiotic-impregnated CaSO 4 beads, as demonstrated through ZOIs., Clinical Significance: When stored correctly, antibiotic-impregnated CaSO 4 beads can be used at least up to 12 months after formulation., (© 2023 American College of Veterinary Surgeons.)