Bottom-up proteomics is a useful approach for the discovery of signature peptides and has been demonstrated to work for bovine milk proteins. Progenesis QI for Proteomics, together with the statistical tools in EZInfo, has streamlined the challenging procedures into an easy-to-use workflow. The signature marker peptides discovered were successfully developed into a quantification analysis, and a range of commercial milk products were analyzed to demonstrate the performance and effectiveness of this method. The automated method created with OneLab, and performed on the Andrew+ Pipetting Robot, greatly reduced the pipetting hours by >5.5 times, while ensuring consistency. This application note illustrated a full suite of solutions and the same workflow can be applied to analyze alternative milk proteins.