Alanine transaminase (ALT) is an important amino acid-metabolizing enzyme in silkwormBombyx moriL., and is mainly involved in transferring glutamate to alanine (serving as an essential precursor in silk protein synthesis) through transamination. Therefore, it is generally believed that silk protein synthesis in the silk gland and the cocoon quantity increase with the increase in ALT activity to a certain extent. Here, a novel analytical method was developed to determine the ALT activity in several key tissues ofBombyx moriL. including the posterior silk gland, midgut, fat body, middle silk gland, trachea and hemolymph, by combining the direct-analysis-in-real-time (DART) ion source with a triple-quadrupole mass spectrometer. In addition, a traditional ALT activity assay, the Reitman–Frankel method, was also used to measure ALT activity for comparison. The ALT activity results obtained via the DART-MS method are in good agreement with those obtained via the Reitman–Frankel method. However, the present DART-MS method provides a more convenient, rapid and environmentally friendly quantitative method for ALT measurement. Especially, this method can also monitor ALT activity in different tissues ofBombyx moriL. in real time.