Recombinant cytochrome P450 (CYP or P450) enzymes are useful for drug metabolism research and thereby many expression and purification systems have been developed. Here, we provide a method for the purification of human P450s 3A4 and 1A2 expressed in Escherichia coli using mixed micelles containing anionic phospholipids. This method does not require any protein-tagging system for protein isolation and has a further advantage that the purification is concomitantly conducted with reconstitution of the enzymes into a phospholipid environment, which is crucial for the catalytic activity assay of P450 enzyme. This method may also be applied to high-throughput catalytic assays of the enzymes because the purification procedures can be undertaken in a 96-well plate. (C) 2014 Elsevier Inc. All rights reserved.