DEqMS is a statistical method to perform differential protein expression (abundance) analysis for quantitative mass spectrometry-based proteomics. Developed on top of Limma, DEqMS implements a robust Bayesian method for accurate variance estimation while taking into account the number of peptide precursors or PSMs used for protein quantification. DEqMS has a demonstrated improved accuracy and statistical power compared to previous methods and has since its initial publication been used by a wide range of researchers in various proteomics applications. Originally, DEqMS was validated to analyze proteomics data from data dependent acquisition (DDA). Based on users’ feedback, we now demonstrate DEqMS usability in performing differential analysis also in data independent acquisition (DIA)-based proteomics data. Given this updated protocol, we envision that DEqMS will continue to be a useful tool for defining sets of proteins with significantly different levels between sample groups based on complex quantitative proteomics data.