The purpose of this study was to establish a reliable and cost-effective microplate proliferation assay for in vitro antimicrobial testing of bone cement samples. Cement samples devoid of antimicrobial agents, loaded with 2% gentamicin or with different concentrations of high-porosity silver, were incubated in a 96-well microplate with several staphylococcal, Pseudomonas aeruginosa, and Enterococcus faecium isolates exhibiting different susceptibilities to gentamicin. After being rinsed, the samples were brought into a soy medium in which adherent cells on the cement surface either were killed by the antimicrobial surface or started to release clonal counterparts. The medium was monitored in real time by recording a time proliferation curve for each well. Microplate testing revealed no antibacterial effect of plain bone cement. The antibacterial activity of gentamicin-loaded bone cement was shown by the microplate test to depend on the gentamicin susceptibilities of the strains. The effect of high-porosity silver was dose dependent. Bactericidal activity against all tested strains was found for bone cement loaded with 1% high-porosity silver. The accuracy of this new proliferation assay was shown by the high correlation between the types of proliferation curves and antibiotic susceptibility. In contrast to routine agar diffusion testing, it assesses the dynamic response of microorganisms to antimicrobial agents in biomaterials and allows high-throughput screening and detection of antimicrobial properties of poorly water-soluble compounds like silver.