Enzyme immunoassay (EIA) and fluorescence polarization immunoassay (FPIA) methods are commercially available for quantitation of serum amikacin concentration. The purpose of this study was to determine if the two assay methods were comparable and would provide the same estimates for pharmacokinetic parameters and dosage recommendations. A total of 73 amikacin serum samples were used to evaluate the two assay techniques. Forty-four of these samples, obtained from 10 patients, were used to evaluate the comparability of pharmacokinetic parameters and dosage regimens. The correlation coefficient between the two assay methods was 0.98 (y= 1.03x+ 0.64). There were substantial differences in assay performance noted in samples < 10 mg/L, 10–20 mg/L, and > 20 mg/L, typical concentration ranges for serum sampling used in pharmacokinetic analysis. A difference of ∼10% was observed in the determination of amikacin half-life, total body clearance, and dosage calculation. A 7% difference was noted in the volume of distribution. A significant difference (p < 0.05) in volume of distribution and dosage recommendations was noted. Although the two methods for determining amikacin serum concentrations appear to be interchangeable on the basis of the in vitro comparison, significant differences were observed between the two assays in pharmacokinetic parameters and dosage recommendations.