The reliability of substrate-labeled fluorescent immunoassay (SLFIA) method was examined using amikacin sulfate (AMK) samples of 5 known concentrations in spiked serum. The result of this SLFIA method were compared with those of enzyme-multiplied immunoassay technique (EMIT), radioimmunoassay (RIA) and bioassay methods. The correlation coefficient of the SLFIA method was 0.9999 and the regression line was Y=1.116X + 0.069. The correlation coefficients of the EMIT, RIA and bioassay methods were more than 0.999. The within-day and between-day coefficients of variation of the SLFIA assay were 2.82-9.13% and 3.04-8.14%, respectively. In AMK-spiked serum samples, the correlation coefficient between the SLFIA and EMIT methods was 0.9996, and the regression line was Y=1.137X-0.181. The value of SLFIA was slightly higher than that of EMIT. Compared with SLFIA and RIA or bioassay, the high correlation was also obtained in AMK-spiked serum samples. In serum of patients undergone AMK therapy, furthermore, the results obtained by SLFIA were compared with those obtained by EMIT, RIA and bioassay. These showed high correlation with each other, though, the value of SLFIA was slightly lower than those of other methods. These results suggest that the SLFIA method may be used for the routine clinical monitoring of serum AMK.