Aims: The antigenic glycopeptidolipids (GPLs) from Mycobacterium avium-intracellulare complex (MAC) are grouped into 28 serovars on the basis of the variable oligosaccharide sequences and the core structures. To facilitate the identification of MAC serovars by employing liquid chromatography/mass spectrometry (LC/MS), the diversity in fatty acyl moieties and the number of acetyl groups of GPLs should be characterized. Methods and Results: Employing a small-scale preparation method, sufficient quantities of intact GPLs could be obtained from several colonies of MAC within 4 h. Tandem mass spectrometry of GPLs showed the presence of common fragment ion at m/z 1048 in the main molecular species of all reference strains. It revealed that the acyl moieties had similar diversity among all serovars. Furthermore, intact GPLs had mainly one or two acetyl groups. This allowed us to determine the masses of each serovar based on intact GPLs and to classify 16 isolates from patients by LC/MS. Conclusions: The present serotyping method using LC/MS analysis improved the precision of measurements and shortened the procedure time compared with conventional thin-layer chromatography or the seroagglutination test method. Significance and Impact of the Study: This proposed method proves useful for identifying serovars of MAC for epidemiological and pathogenic research purposes.