Mass spectrometry-based proteomics and metaproteomics have long been used in the study of human microbiomes, with the potential of metaproteomics only recently being fully harnessed. This progress is due to the advancements of high-performance mass spectrometers, innovative proteomics strategies, and the development of dedicated bioinformatics tools. In this review, we critically examine the recent technological developments that enhance the application of metaproteomics in clinical microbiome analysis. We also summarize significant advancements in the application of metaproteomics to study human microbiomes across various body sites under disease conditions. Despite these, the potential of metaproteomics remains underutilized due to typically small sample sizes and insufficient data mining. We thereby highlight some key aspects that could facilitate the broader and more effective application of mass spectrometry-based metaproteomics in clinical microbiome analysis, including the development of microbiome assays for translational research and application.