MSRefine is a tool for extracting comprehensive metrics and assisting users in evaluating and optimizing the performance of LC–MS/MS. This tool extracts dozens of detailed metrics that cover a wide range of information, including sample preparation, liquid chromatography separation and mass spectrometry data acquisition. It also enables the analysis of vendor data generated from Thermo Orbitrap, Bruker timsTOF Pro and SCIEX ZenoTOF instruments. More importantly, we established the relationship between the extracted metrics and the identification results through a scoring system in MSRefine for the DDA workflow, directly and effectively indicating more appropriate strategies for deeper proteome profiling. MSRefine also outputs a report that contains various graphs or tables, such as radar graphs, to give a global view of the performance of the experiment. For different kinds of samples and instruments, a set of optimal parameters can also be quickly determined to identify more peptides and proteins according to the scoring system and comprehensive reports. Altogether, these features make MSRefine a valuable software tool for evaluating and optimizing the performance of experiments for deeper proteomics.