Lipid profiling in nine bacterial species has been accomplished by laser desorption ionization mass spectrometry (LDI-MS) using amorphous silicon (a-Si) thin film with 100 nm thickness. Lipid ions could be generated by LDI on a-Si regardless of ion acquisition modes because of a thermal property of a-Si to govern laser-induced surface heating. In a comparative study of lipid profiling in Bacillus lichemiformis by LDI-MS and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), LDI-MS on a-Si shows a higher efficiency in lipid and lipopeptide detection than MALDI-MS. A total of 53 peaks of lipid ions generated by LDI on a-Si in both acquisition modes for m/z 400-1200 was 1.6 times more than that detected by MALDI-MS using three organic matrices-2,5-dihydroxybenzoic acid, 1,5-diaminonaphthalene, and 2,4,6-trihydroxyacetophenone monohydrate. Also, the authors demonstrate by mass spectrometry imaging (MSI) that LDI-MS provides high detection coverage through whole sample area. MSI results show the detection yield in LDI on a-Si is 94.8% calculated by counting the number of points detected in the analyte ion signal in a whole spot. It means that reproducible detection of lipid ions by LDI-MS is possible even if laser is randomly irradiated at any position within the bacterial sample area applied on a-Si. Lipid profiling by LDI-MS on a-Si was applied to bacterial differentiation of nine bacterial species conducted by performing principal component analysis. Nine bacterial species are successfully distinguishable from each other by LDI-MS lipid profiling.