An in vitro cell-based bioassay capable of detecting neutralizing antibodies (NAb) to recombinant human erythropoietin (rHuEPO) in clinical samples was developed and validated. The bioassay uses the IL-3-dependent murine 32D cell line transfected with human EPO receptors (EPOR). This cell line responds to rHuEPO with proliferation measurable by [methyl-3H] thymidine incorporation into the cellular DNA. The reduction of rHuEPO-induced cell proliferation response indicates the possible presence of anti-rHuEPO NAb. In addition, a specificity assay using murine IL-3 (mIL-3) induced proliferation of the same cell line was developed and validated. The specificity assay allowed testing of samples that inhibited the biologic activity of rHuEPO to evaluate whether the inhibition was specific and not attributable to cytotoxicity of the serum sample. Both assays are conducted in a 5% human serum matrix in 96-well microtiter plates. The Guidelines of the International Conference on Harmonization (ICH) were followed for the validation of different assay parameters including analytical recovery, precision, sensitivity, specificity, selectivity, and robustness. The anti-rHuEPO NAb assay is capable of detecting concentrations of NAb equivalent to 500 ng/ml of the positive control antibody in undiluted human serum. The anti-rHuEPO NAb assay yielded consistent results with cells cultured for up to 30 days. The positive control antibody maintained its ability to inhibit the biologic activity of rHuEPO upon freezing and thawing. The presence of free rHuEPO in serum samples interfered with the detection of the antibody. The validated assay was sensitive, specific and robust and was successfully used to monitor NAb development in patients.