Whey protein, an essential component of milk, possesses a rich repertoire of bioactive proteins with unique nutritional and functional properties. Employing liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), we conducted a comparative analysis of whey proteins derived from enzyme curd Esprion300HS (E300HS) and acid curd Textrion PROGEL 800 (TP800) methods. Our investigation identified 216 proteins, of which 32 exhibited differential expression between E300HS and TP800. Notably, E300HS displayed significantly higher levels of calcium-binding protein, lactoperoxidase, and lactadherin compared to TP800. Peptide profiling revealed 162 peptides, with 12 upregulated and 14 downregulated in E300HS relative to TP800. Furthermore, E300HS exhibited abundant lactoferrin-derived peptides, while TP800 displayed a higher prevalence of casein-like peptides. These proteins and peptides encompass a broad range of biological functions, including anticancer, antimicrobial, and bone remodeling activities. Our findings underscore the vast potential of whey protein in food industry applications and its significance in cancer research, bone development, and age-related degenerative diseases.