Digest of proteins in gel or in solution is one of the key steps of protein, peptide and modified amino acids identification by LCMSMS. There is always a need to improve speed and quality of digest. Pressure assisted technologies were shown to improve the digest in solution. However, there are practically no studies on pressure assisted digest in-gel, which could yield more or complimentary peptides, enhancing peptide/protein identifications•Digest of proteins in gel or in solution is one of the key steps of protein, peptide and modified amino acids identification by LCMSMS. There is always a need to improve speed and quality of digest. Pressure assisted technologies were shown to improve the digest in solution. However, there are practically no studies on pressure assisted digest in-gel, which could yield more or complimentary peptides, enhancing peptide/protein identifications•Here we report results on optimization of in-gel tryptic digestion using pressure-assist technology in a Barocyclerat different temperatures/ pressures 37C/20,000 psi, 37C/45000 psi, 50C/45000 psi and comparison with conventional atmospheric pressure tryptic in-gel digestion.