Detection of species-specific proteotypic peptides for accurate and easy characterization of infectious non-tuberculous mycobacteria such as Mycobacterium smegmatis is essential. Therefore, we carried out reanalysis of publicly available M. smegmatis proteomic dataset PXD003303, PXD004197, PXD010020, PXD016241, PXD017602, PXD027848, PXD027855 and PXD029588 by proteome database search and followed by spectral library generation. The raw DDA data were searched against their respective reference proteome databases using Proteome Discoverer and FragPipe. The resulting peptide spectrum matches were converted into a spectral library using BiblioSpec.