Milk represents an integrated meal for newborns; its whey protein is rich in many health beneficial components and proteins. The current study aimed to investigate the differences between colostrum and mature milk from Mediterranean and Murrah buffaloes using labeled proteomics and bioinformatics tools. In the current work, LC-MS/MS analysis led to identification of 780 proteins from which 638 were shared among three independent TMT experiments. The significantly changed proteins between the studied types were analyzed using gene ontology enrichment and KEGG pathways, and their interactions were generated using STRING database. Results indicated that immunological, muscular development and function, blood coagulation, heme related, neuronal, translation, metabolic process, and binding proteins were the main terms. Overall, colostrum showed higher levels of immunoglobulins, myosins, actin, neurofascin, syntaxins, thyroglobulins, and RNA-binding proteins, reflecting its importance in the development and activity of immunological, muscular, cardiac, neuronal, and thyroid systems, while lactoferrin and ferritin were increased in mature milk, highlighting its role in iron storage and hemoglobin formation