For prosthetic joint infections, antibiotic loaded poly methyl methacrylate (PMMA) spacer or beads can be used to release high concentrations of antibiotics locally at the infection site, while minimizing systemic toxicity. The aim of this study is to determine in vitro and in vivo pharmacokinetic release profile of antibiotics from PMMA spacers and PMMA beads. For the in vitro experiment, the PMMA spacers or beads were submerged in phosphate-buffered saline and gentamicin concentrations were determined from collected specimen at several times points, measured with enzyme-linked immunosorbent assays (ELISA). To assess the in vivo antibiotic release profile of different spacers, wound drainage fluid samples were collected after implantation of a spacer over a period of maximum 14 days. After 48 h, the burst gentamicin concentration elution was 9862 ± 1782 ng/ml (mean ± SD) from spacers versus 38,394 ± 7071 ng/ml (mean ± SD) for beads. Over 35 days, spacers had eluted a cumulative mean concentration of 13,812 ± 3548 versus 55,048 ± 12,006 ng/ml for beads (p < 0.001). Clinical samples of patients with a Vancogenx® spacer showed higher gentamicin release than Refobacin™ spacers (p < 0.001). This is the first study that measured the release data of local antibiotics with ELISA. Compare to spacers, the exact release values of gentamicin from PMMA beads are more than 10 times higher and reached a maximum much later than spacers. This makes the use of PMMA beads more preferable to use for treatment of the infection itself.