We developed an Optimized Salivary MetaProteomic sample analysis workflow (O-SaMPle) to enrich salivary bacteria and reduce host-derived interferences for in-depth analysis of the oral metaproteome. This workflow integrates using selective microbial enrichment buffer and differential centrifugation. In details, the selective microbial enrichment buffer can dissolves host proteins and detaches microorganisms from cells while preserving the integrity of the microbes by using a combination of low concentrations of denaturing agents and detergents, followed differential centrifugation can effectively isolate and enrich oral microbiota from human saliva or mouth rinse samples. Furthermore, we applied O-SaMPle to analyzing mouth rinse samples from IBD patients, and revealed dysregulated microbes and their corresponding dysregulated pathways under disease conditions.