To describe a rapid and precise protocol for peptide analysis utilizing immobilized, heat stable trypsin and micro-elution solid-phase extraction (SPE) coupled with next generation ultra-high pressure liquid chromatography and high resolution mass spectrometry (UHPLC-HRMS). Traditional trypsin digest protocols are time consuming, with some protocols taking up to one and a half days to complete and involving multiple steps including evaporation and reconstitution. This adds potential for a high degree of variability. The Thermo Scientific™ SMART Digest™ kit was used to digest cytochrome C with a 10 minute digestion protocol. This was followed by Thermo Scientific™ SOLAμ™ SPE plates to clean and concentrate the digest prior to injection onto the Thermo Scientific™ Vanquish™ UHPLC system. Following separation on a Thermo Scientific™ Acclaim™ C18 RSLC analytical column, high-resolution mass spectrometry was performed using a Thermo Scientific™ Q Exactive™ Plus hybrid quadrupole-Orbitrap mass spectrometer. A high-throughput, reproducible workflow was achieved that can be applied to nontargeted, semitargeted, or targeted quantitative environment for peptide analysis.