Abstract: OBJECTIVE To compare enzyme-multiplied immunoassay technique(EMIT) and fluorescence polarization immunoassay(FPIA) method in the determination of valproic acid(VPA), methotrexate(MTX) and cyclosporine A(CsA) concentrations in human plasma, and to evaluate the correlation of concentrations determined by EMIT and FPIA. METHODS Plasma samples were collected from patients. Both EMIT and FPIA method were employed to determine the concentrations of VPA, MTX and CsA. Regression analyses were performed (EMIT results as dependant, FPIA as independent variable) for the evaluation of correlation. RESULTS The regression equation of the results determined by FPIA (X) and EMIT (Y) were Y_VPA=-1.894 1+1.190 3X, r=0.983; Y_MTX=0.099 24+1.136X, r=0.992; Y_CsA=-1.146 5+0.846 1X, r=0.971. VPA concentration determined by EMIT was 11.2 μg·mL^-1 higher than FPIA. MTX concentration determined by EMIT was 0.22 μmol·L^-1 higher than FPIA. CsA concentration determined by EMIT was 20.6 ng·mL^-1 lower than FPIA(P<0.05). CONCLUSION Attentions should be paid to the differences of VPA, MTX and CsA concentration determined by EMIT and FPIA methods in dosage modification.