Updated publication reference for PubMed record(s): 33320415. Using Mycoplasma pneumoniae as a model organism, we conditionally depleted the two essential ATP-dependent proteases (Lon and FtsH) of this bacterium, by engineering strains carrying a Lon and/or FtsH inducible expression locus. An integrative comparative study combining label-free shotgun proteomics and RNA-seq allowed us to decipher the global cellular response to Lon and FtsH depletion and to define protease substrates in this genome-reduced organism. This particular dataset corresponds to the analysis of the Triton X-100 insoluble fractions of Lon and FtsH mutants grown under inducing and depleting conditions. The dataset for the whole cell lysate is available under the identifier PXD016343.