A simple and reproducible method for extraction of membrane enzymes from Gram positive bacteria in active form has been developed. The method employs the incubation of cells with lysozyme and sonication in the presence of salt and detergent followed by repeated freezing and thawing to disrupt the lipid–protein interactions. Although developed for Dipeptidylpeptidase-III as a demonstration, this protocol has been used for the successful extraction of some synthetic substrate hydrolyzing activities from three different Gram positive bacteria with yield of more than 90% for most studied activities. This method can be routinely used for a wide range of applications and it can also be used to extract and solubilize other enzymes/proteins only by manipulating detergent, salt and their concentration. It is simple, straightforward, reproducible, cost effective and a less time consuming protocol for extracting and solubilizing membrane enzymes.