Abstract: The experimental conditions of matrixassisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) in the analysis of proteins and glycoproteins were studied and were used to analyze the molecular weights and purity of nine recombinant bioactive proteins, such as interleukin 2, tumor necrosis factor α, granulocytemacrophage colony stimulating factor, interferon α_2b, interferon α₁, erythropoietin, calmodulin and its fragments, neural nitric oxide synthase, and a natural protein extracted from bovine semen. It was also applied to determine the objective protein and calmodulin in an unknown mixture. In addition, the single ingredient in protein mixtures was characterized by the MALDI-TOF MS. The results showed that the technique of MALDI-TOF MS can be employed to determine the quality of recombinant and natural proteins effectively and accurately.