This review provides an updated overview of the subtilisin-like serine proteases, known as subtilases, which are a superfamily of over 200 members, with more than 170 having complete amino acid sequences. The authors summarize over 100 new subtilases discovered in the past five years and compare their catalytic domain sequences using multiple sequence alignment. Based on sequence homology, the subtilases are subdivided into six families: subtilisin, thermitase, proteinase K, lantibiotic peptidase, kexin, and pyrolysin. Highly conserved residues in the catalytic domain are identified, along with large or unusual deletions and insertions. Predictions are made for Ca²⁺-binding sites, disulfide bonds, and substrate specificity, based on sequence alignment and three-dimensional homology modeling. The review also discusses the structure-based alignment of subtilases, the identification of new subtilase superfamily members, and the sequence homology and family division. The authors highlight the high degree of sequence variability within the catalytic domains and the need for more detailed three-dimensional structural information to complement sequence data. They conclude by noting the ubiquitous presence of subtilases in Archaea, Bacteria, and Eucarya, and their diverse functions, particularly in higher eukaryotes where they are involved in biosynthesis pathways.This review provides an updated overview of the subtilisin-like serine proteases, known as subtilases, which are a superfamily of over 200 members, with more than 170 having complete amino acid sequences. The authors summarize over 100 new subtilases discovered in the past five years and compare their catalytic domain sequences using multiple sequence alignment. Based on sequence homology, the subtilases are subdivided into six families: subtilisin, thermitase, proteinase K, lantibiotic peptidase, kexin, and pyrolysin. Highly conserved residues in the catalytic domain are identified, along with large or unusual deletions and insertions. Predictions are made for Ca²⁺-binding sites, disulfide bonds, and substrate specificity, based on sequence alignment and three-dimensional homology modeling. The review also discusses the structure-based alignment of subtilases, the identification of new subtilase superfamily members, and the sequence homology and family division. The authors highlight the high degree of sequence variability within the catalytic domains and the need for more detailed three-dimensional structural information to complement sequence data. They conclude by noting the ubiquitous presence of subtilases in Archaea, Bacteria, and Eucarya, and their diverse functions, particularly in higher eukaryotes where they are involved in biosynthesis pathways.