In this study, we have prepared a hydrophobic cryogel for the chromatographic separation of lipase from its aqueous solutions including single protein and protein mixture and also Yarrowia lipolytica cell extract. N-methacryloyl-(l)-phenylalanine methyl ester was used as a monomer to provide the hydrophobic character to the prepared cryogels. The highest adsorption capacity was observed at pH 5.0 at 0.5 mL min(-1) flow rate. The chromatographic separation of lipase was achieved from a binary mixture of lipase:bovine serum albumin (BSA) and lipase:lysozyme, and was also achieved from triple-mixture of lipase:lysozyme:BSA by using fast protein liquid chromatography. Finally, lipase purification was performed from Yarrowia lipolytica cell extract used as a natural source. These studies have shown that the hydrophobic cryogel has good chromatographic performance for the separation and purification of lipase not only from aqueous solution, but also from cell extract as a natural source of lipase. (c) 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:376-382, 2014