The importance of the pathogenic mycobacteria has mainly focused the omic analyses on different aspects of their clinical significance. However, those industrially relevant mycobacteria have received less attention, even though the steroid market sales in 2021 were estimated in $56.45 billion.The extracellular proteome, due to its relevance in the sterol processing and uptake, and the intracellular proteome, because of its role in steroids bioconversion, are the core of the present chapter. Both, monodimensional gels, as preparatory analysis, and bidimensional gels as proteome analysis are described. As a proof of concept, the protein extraction methods for both sub-proteomes of Mycobacterium are described. Thus, procedures and relevant key points of these proteome analyses are fully detailed.