Recent literature has urged caution in the interpretation of vancomycin serum concentrations in patients with end-stage renal disease (ESRD), because falsely elevated levels in excess of 70% have been reported with the most commonly used fluorescence polarization immunoassay (FPIA). The purpose of this study was to evaluate the performance of a recently modified FPIA assay for use in patients with ESRD, in comparison to high-performance liquid chromatography (HPLC) and an enzyme-mediated immunoassay technique (EMIT). Serum vancomycin samples were prospectively collected from adults with ESRD undergoing chronic hemodialysis. Each sample was stored at -70°C until analyzed in duplicate by FPIA, EMIT, and HPLC. In an in vitro experiment, blank serum samples with 15 µg/ml vancomycin were spiked with increasing amounts of CDP and analyzed in duplicate with the modified FPIA assay. When compared to HPLC, no statistically significant difference was found in patients with ESRD with the use of the modified FPIA assay (mean concentrations, HPLC 14.92 µg/ml, FPIA 15.96 µg/ml), with FPIA exhibiting a positive bias of 0.64 µg/ml and a precision of±3.49 µg/ml (n = 18, p = 0.44). The mean EMIT concentration was 18.34 µg/ml, with a positive bias of 3.43µg/ml and a precision of ±5.17 µg/ml (p < 0.01). The addition of increasing amounts of CDP to vancomycin in vitro resulted in concentrations similar to those expected in the absence of significant cross-reactivity with the modified FPIA assay. The modified FPIA assay is a satisfactory tool for monitoring vancomycin serum concentrations in patients with ESRD undergoing hemodialysis. Results obtained with EMIT were not as precise as with FPIA.