The ability of tandem mass spectrometry to determine the primary structure of proteolytic peptides can be exploited to trace back the organisms from which the corresponding proteins were extracted. This information can be important when food products, such as protein powders, can be supplemented with lower-quality starting materials. In order to dissect the origin of proteinaceous material composing a given unknown mixture, a two-step database search strategy for bottom-up nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) data was implemented. A single nanoLC-MS/MS analysis was sufficient not only to determine the qualitative composition of the mixtures under examination, but also to assess the relative percent composition of the various proteomes, if dedicated calibration curves were previously generated. The approach of two-step database search for qualitative analysis and proteome total ion current (pTIC) calculation for quantitative analysis was applied to several binary and ternary mixtures which mimic the composition of milk replacers typically used in calf feeding.