Fractionation of bovine milk was performed using chymosin-induced separation, isoelectric precipitation or ultracentrifugation as separation techniques prior to gel-based proteomic analysis. This approach allowed for comparative display and identification of proteins partitioned into casein and whey, respectively. Initially, three different staining methods (silver staining, colloidal Coomassie Blue G-250 or fluorescent Flamingo Pink staining) for two-dimensional gel electrophoresis (2-DGE) analysis were compared for their suitability as staining agent, especially in relation to their suitability to reveal differences in the casein fractions. Fluorescent staining proved to be the most appropriate for this purpose, giving a high sensitivity, and using this staining method, characteristic 2-DGE fingerprints were obtained for each casein and whey fraction from each separation method. A number of protein spots in both casein and whey fractions varied with separation method and these spots were subsequently identified using tandem mass spectrometry (MS). In rennet casein, proteolytic fragmentation of caseins (αs1-, αs2,-, β- and κ-) was identified as a result of chymosin hydrolysis, whereas the 2-DGE profile of acid and ultracentrifuged casein was dominated by the presence of multiple isoforms of κ-caseins. Furthermore, casein remnants were identified in milk serum after ultracentrifugation. This study shows that gel-based proteomic analysis is suitable for characterisation of subtle variations in protein composition of milk fractions that occur as a consequence of different milk fractionation strategies.