A method for simultaneous analysis of 16 free amino acids in entomogeneous fungi was developed using liquid chromatography-electrospray time-of-flight mass spectrometry(LC-ESI-TOF-MS) under negative ionization mode.The results showed that chromatographies and ion features of the 16 amino acids had obvious characteristics which can be used for accurately qualitative analysis.Quantitative tests showed that the 16 amino acids had wide linear ranges and high accuracy.In the linear range from 4.0 to 125μmol/L all the correlation coefficients between concentration and peak area of the 16 amino acid were above 0.998.At the concentrations of 50μmol/L the relative standard deviations of the 16 amino acids were in the range from 0.84% to 2.75%.Results of LC-ESI-TOF-MS analysis of the 16 free amino acids in mycelia of 7 kinds of entomogenous fungi showed that the contents of the 16 amino acids were very different in the same strain.Of all the amino acids the content of phenylalanine was the highest one in all tested strains,and its range was from 0.718 to 2.173 mg/g.Hirsutella sinensis contained the highest phenylalanine.Apart from phenylalanine the contents of proline,valine,leucine,isoleucine,lysine and glutamic acid were also obviously higher than the other amino acids,and most of them were higher than 0.3mg/g.The amino acids with relative higher contents could be used as characteristic index for identification of free amino acids in entomogenous fungi.The total free amino acids contents in mycelia of different strains were also significantly different.The content of Hirsutella sinensis was 7.537mg/g which was 1.36 times higher than that of Isaria cicadae(3.196mg/g).