Established bacterial proteome sample preparations, including sonication and bead beating, leave insoluble carbohydrate-rich cell envelope pellets with an abundance of vital proteins often overlooked or missed in LC-MS/MS analyses. Triflic acid selectively removes glycans and we demonstrate that in comparison to sonication alone, incubation of whole bacterial cells as well as post-sonication insoluble pellets yields membrane and cell envelope-associated proteins for LC-MS/MS detection. We provide a detailed side-by-side comparison of triflic acid and sonication preparations for Gram- (Pseudomonas aeruginosa), Gram+ (Bacillus subtilis), and a complex bacterial human distal gut microbiome sample. Further, human Jurkat cells that lack a peptidoglycan and are readily solubilized by established methods, reveal only subtle differences in measurable proteins by LC-MS/MS between sonication and triflic acid preparations. Critically, we show that our new triflic acid-based proteome preparation method is broadly applicable and greatly improves our ability to detect and quantitate bacterial cell envelope proteins.