High-throughput screening (HTS) is a key step to the success of directed evolution of enzymes. Recently, fluorescence-activated cell sorting (FACS) has emerged as a promising tool for HTS. Directed evolution of Fusarium solani cutinase, which is partially released from E. coli cells, was carried out using a FACS-based screening technique to increase its activity for (R)-flurbiprofen. First, the ability of using the FACS-based screening technique to screen active cutinase using wild type cutinase (WT) and inactivated cutinase mutant (S42A) was examined. Although the FACS-based screening using E. coli cells did not work well due to the diffusion of fluorescent product and/or an interference by the partially released cutinase from the the cells, FACS could be used to effectively screen active wild type cutinase via in vitro compartmentalization using water/oil/water emulsion microcompartments. Cutinase variants showing higher activity for (R)-flurbiprofen could be screened after four screening steps. The mutants 2-95 and 0-5 showed 8.0- and 6.8-fold higher activities for fluorescein (R)-flurbiprofen diester compared to that of the wild type cutinase, respectively. The mutant 0-5 also showed 5.0-fold higher activities for fluorescein butyl diester compared to that of the wild type cutinase.