More than 560 genes are annotated as proteases in the human genome. About half of the genes are not or are only marginally characterized. Over the past decade, mass spectrometry has become the basis for proteomics, especially for protein identification, performed in a high-throughput manner. This development was also very fruitful for exploring the complex systems associated with protease functions, as briefly reviewed here. Mass spectrometry is an ideal tool for monitoring protease reactions, as will be highlighted in this review.