PROSITE is a comprehensive resource for identifying and annotating conserved regions in protein sequences, including protein families, domains, and motifs. The latest version (release 20.85) includes 1308 patterns, 1039 profiles, and 1041 ProRules. Recent developments have enhanced the utility of the ScanProsite tool, allowing users to perform whole-proteome annotation and targeted searches with filtering options. Users can upload complete proteome sets for analysis, and the tool supports combinatorial searches using logical operators to combine signatures. Filtering options include taxonomic classification, protein names, tissue expression, and size constraints. These features enable users to integrate prior biological knowledge with sequence features for powerful targeted searches. An example is provided using the alkylglycerol mono-oxygenase of *M. musculus*, where ScanProsite helped identify candidate sequences for experimental validation. Future improvements aim to enhance the speed of ScanProsite for better proteome annotation.PROSITE is a comprehensive resource for identifying and annotating conserved regions in protein sequences, including protein families, domains, and motifs. The latest version (release 20.85) includes 1308 patterns, 1039 profiles, and 1041 ProRules. Recent developments have enhanced the utility of the ScanProsite tool, allowing users to perform whole-proteome annotation and targeted searches with filtering options. Users can upload complete proteome sets for analysis, and the tool supports combinatorial searches using logical operators to combine signatures. Filtering options include taxonomic classification, protein names, tissue expression, and size constraints. These features enable users to integrate prior biological knowledge with sequence features for powerful targeted searches. An example is provided using the alkylglycerol mono-oxygenase of *M. musculus*, where ScanProsite helped identify candidate sequences for experimental validation. Future improvements aim to enhance the speed of ScanProsite for better proteome annotation.