Background: In the setting of prosthetic joint infections treated with a two-stage procedure, spacers can be sonicated after removal. We hypothesize that the sonication process may cause an increased elution of antibiotics from the spacer, leading to elevated concentrations of antibiotics in the sonication fluid inhibiting bacterial growth. We aimed to evaluate in vitro the influence of sonication on the elution of antibiotics from polymethyl methacrylate (PMMA) over time and to determine whether these concentrations are above the minimum inhibitory concentrations (MIC) for microorganisms relevant in prosthetic joint infections. Methods: PMMA blocks impregnated with vancomycin, fosfomycin, gentamicin or daptomycin were incubated in phosphate-buffered saline (PBS) at 37°C for up to 6 weeks. PBS was changed once a week. Concentrations were determined from samples of each antibiotic every week, and after 5 minutes of sonication at 2, 4 and 6 weeks. Results: With sonication there was a trend toward an increase of the elution of antibiotics. This increase was significant for vancomycin at 2 and 4 weeks (p=0.008 and 0.002 respectively) and for fosfomycin at 2 weeks (p=0.01). Conclusion: The effect of sonication could play a role in clinical results, especially for daptomycin and gentamicin for which the MIC is close to the concentration of antibiotics at 4 and 6 weeks. We conclude that elution of antibiotics from PMMA along with the effect of sonication could inhibit bacterial growth from spacers, resulting in false negative results in the setting of two-stage exchange procedures for prosthetic joint infections.