We developed a method to evaluate the contents of serum protein fractions using nonlinear least squares approach. Parameters that determined the distributions of fractions of a densitogram were estimated assuming that each fraction obeys Gaussian distribution. One hundred test results of patients and one hundred and fourteen simulated patterns were analyzed with the proposed method and the traditional method. For patient data, the traditional method tended to give higher values in beta-globulin fraction and lower values in alpha 2-globulin and gamma-globulin fractions compared with our method. For the simulated data, the traditional method also tended to give higher values in beta-globulin fraction and lower values in gamma-globulin fraction, whereas our method gave the correct values.