Saliva gained interest as a potential noninvasive source of biomarkers in humans and that interest starts to be extended also to other animal species. For this purpose, the knowledge of the salivary proteome in healthy conditions and the factors that affect it and how they affect it are necessary. The aim of the present study was to assess the effect that gender and breed have in saliva proteome and the changes in it induced by stimulation with acid. Saliva from 4 different purebred dogs (Portuguese Podengo, Greyhound, Rafeiro Alentejano, and Beagle) of both genders was collected without and after stimulation with lemon juice. SDS-PAGE and two-dimensional gel electrophoresis (2-DE) profiles were compared and the proteins of interest in-gel digested and identified by mass spectrometry. Acid stimulation decreased total protein concentration and the relative amounts of some protein bands/spots. Gender appeared to have minimal effect in saliva proteome, whereas the influence of breed varies. Beagles and Portuguese Podengos were the two breeds with higher differences. In conclusion, stimulation procedures and dog breed should be considered in data analysis when using salivary proteins for diagnostic purposes.