Rifamycins are a class of antibiotic compounds of which rifampicin is the most commonly prescribed. Conventional electron-impact mass spectrometry of rifampicin has not been found to provide useful data. Thermospray and electrospray mass spectrometry are studied as potential tools for the analysis of rifampicin, rifamycin SV, and rifamycin B. Using thermospray and electrospray ionization, all three compounds provide significant ion intensity for either the [MH]+ or [MNa]+ ions. In addition, combined high-performance liquid chromatography-thermospray mass spectrometry provides useful analytical data for a mixture of the three rifamycins.