The use of saliva as a protein source prior to microbiological and biological assays requires previous processing. However, the effect of these processing methods on the proteomic profile of saliva has not been tested. Stimulated human saliva was collected from eight healthy volunteers. Non-processed saliva was compared with 0.22 μm filtered, 0.45 μm filtered, and pasteurized saliva, by liquid chromatography−mass spectrometry. Data are available via ProteomeXchange with identifier PXD039248. The effect of processed saliva on microbial adhesion was tested using bacterial and fungus species and in biological cell behavior using HaCaT immortalized human keratinocytes. Two hundred and seventy-eight proteins were identified in non-processed saliva, of which 54 proteins (≈19%) were exclusive. Saliva processing reduced identified proteins to 222 (≈80%) for the 0.22 μm group, 219 (≈79%) for the 0.45 μm group, and 201 (≈72%) for the pasteurized saliva, compared to non-processed saliva. The proteomic profile showed similar molecular functions and biological processes. The different saliva processing methods did not alter microbial adhesion (ANOVA, p > 0.05). Interestingly, pasteurized saliva reduced keratinocyte cell viability. Saliva processing methods tested reduced the proteomic profile diversity of saliva but maintained similar molecular functions and biological processes, not interfering with microbial adhesion and cell viability, except for pasteurization, which reduced cell viability.