Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based microbial identification is a popular analytical method. Strain Solution proteotyping software available for MALDI-TOF MS has great potential for the precise and detailed discrimination of microorganisms at serotype- or strain-level, beyond the conventional mass fingerprinting approaches. Here, we constructed a theoretically calculated mass database of Salmonella enterica subspecies enterica consisting of 12 biomarker proteins: ribosomal proteins S8, L15, L17, L21, L25, and S7, Mn-cofactor-containing superoxide dismutase (SodA), peptidyl-prolyl cis-trans isomerase C (PPIase C), and protein Gns, and uncharacterized proteins YibT, YaiA, and YciF, that can allow serotyping of Salmonella. Strain Solution ver. 2 software with the novel database constructed in this study demonstrated that 109 strains (94%), including the major outbreak-associated serotypes, Enteritidis, Typhimurium, and Infantis, could be correctly identified from others by colony-directed MALDI-TOF MS using 116 strains belonging to 23 kinds of typed and untyped serotypes of S. enterica from culture collections, patients, and foods. We conclude that Strain Solution ver. 2 software integrated with the accurate mass database will be useful for the bacterial proteotyping by MALDI-TOF MS-based microbial classification in the clinical and food safety fields.