Chiral stationary phases based on human serum albumin were prepared by using a reactive polymer, polyacryloyl chloride, to attach the protein to the silica particles. The retention data of the enantiomers depend on the structure of the polymer sublayer. In most cases, higher selectivity values are observed when the polymer coating is formed by polymerization inside the pores of the silica (in situ polymerization) than with polymer layers deposited by conventional coating methods. This result was attributed to the higher mobility of polymer chains obtained by the former method, which results in lower distortion of the protein after immobilization.