The aim of the study was to investigate the possibility to differentiate the 4 most important species in Italian dairy industry (cow, buffalo, sheep, and goat), applying a bottom-up proteomic approach to assess the milk species involved in cheese production. Selective peptides were detected in milk to use as markers in cheese products. Trypsin-digested milk samples of cow, sheep, goat, and buffalo, analyzed by HPLC-tandem mass spectrometry provided species-specific peptides, some of them recognized by Mascot software (Matrix Science Ltd., Boston, MA) as derived from well-known species specific proteins. A multianalyte multiple reaction monitoring method, built with these specific peptides, was successfully applied to cheeses with different composition, showing high specificity in detection of species involved. Neither aging nor production method seemed to affect the response, demonstrating that chosen peptides well act as species markers for dairy products.