Background: Orthopaedic implant infections are treated by surgical debridement, systematic antibiotic treatment or local antibiotic treatment with antibiotic-loaded beads. Currently antibiotic concentrations in wound exudate, serum, urine or tissue samples are determined with HPLC or fluorescent spectrometric assays. Both methods are heavily influenced due to proteins in the samples. Questions/purposes: Is ELISA capable to detect gentamicin and vancomycin in protein-containing samples like serum and wound exudate. Methods: Two specific competitive ELISA-assays were set-up to detect either gentamicin or vancomycin in protein-rich samples. An antibiotic-BSA hapten was generated as a coatable antigen and commercially available antibodies were applied for downstream immunodetection. Results: The developed ELISAs perform at a detection range of 2–500 ng/ml gentamycin and 20–5000 ng/ml vancomycin. Both ELISAs were capable of detecting these antibiotics in human serum and wound exudate without being compromised by the presence of proteins. We did not detect cross-reactivity for gentamicin in the vancomycin ELISA or vice versa. Conclusions: The antibiotic ELISAs detect gentamicin and vancomycin at low concentrations in protein-rich samples and they can be used as a high throughput and cost-effective alternative for chromatographic or fluorescent methods. Clinical relevance: These ELISAs can be used to detect very low gentamicin or vancomycin concentrations in clinical samples or assess novel orthopaedic antibiotic release systems in in vitro and in vivo studies.