The single-molecule PCR-linked in vitro expression (SIMPLEX) technology, which can directly link a single molecule of a gene to its encoding protein, has been used to engineer enantioselectivity of lipase from Burkhorderia cepacia KWI-56. A combinatorial mutation has been introduced only to four residues in the hydrophobic substrate-binding pocket of the enzyme based on a structural model of the substrate-enzyme complex. Such focused mutation library constructed by the SIMPLEX technology has been screened for an enantiomeric substrate. Some combinations of substitutions in the four positions of the lipase have been found as effective for changing the enantio-preference from the (S)-form of p-nitrophenyl-3-phenylbutyrate to the (R)-form. Here, we describe the detail procedure to construct such an exclusively in vitro protein library and a practical screening method based on enzymatic activity.