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.