Owing to the wide substrate specificity and higher expression levels in recombinant host, these lipases have tremendous importance for hydrolysis and synthesis reactions. Various substrates with substitutions on the alcohol and/or the acid part of the ester molecule were selected. The experimental results support the classification of lipases on the basis of their binding sites. For substrates with heavy alcohol side, C. Antarctica lipase A and R. miehei lipase type enzymes gave the highest extent of hydrolysis, while for acid heavy substrates the highest conversions were shown by C. antarctica lipase B. It is noteworthy that the acid heavy substrates which had aromatic side chains were hydrolyzed only by C. antarctica lipase B type of enzymes. Lipases were found to be more active on the alcohol-substituted substrates than acid-substituted substrates.