Updated project metadata. The in-gel digestion of proteins for analysis by liquid chromatograph mass spectrometry has been used since the early 1990s. Although several improvements have contributed to increasing the quality of the data obtained, many recent publications still use sub-optimal approaches. We present an updated in-gel digestion protocol. We show that alternative reducing, alkylating agents and tryptic digestion buffers increase peptide and protein identification and reduce incubation times. Our results indicate that a simultaneous and short, high temperature reduction and alkylation reaction using Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and chloroacetamide (CAA) with a subsequent gel wash improve protein identification and sequence coverage, diminish peptide side reactions. Additionally, use of 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid buffer (HEPES) allows a significant reduction in the digestion time improving trypsin performance and increasing the peptide recovery. The updated in-gel digestion protocol described here is highly efficient and offers flexibility to be incorporated in any proteomic laboratory.