Screening assays are the most time-consuming and labor-intensive part of generating monoclonal antibodies (MAbs). Antibodies identified by enzyme-linked immunosorbent assay (ELISA) screening often are not suitable for their intended application such as immunofluorescence staining. We describe here a rapid and efficient flow cytometric screening procedure for the identification of MAbs directed against low-abundance cytoplasmic proteins, in our case, the pro-apoptotic molecule Bim. Cells from an equal mixture of a parental cell line and a subline expressing Bim were fixed, permeabilized and incubated with hybridoma supernatants. The supernatants were derived from a fusion of Sp2/0 plasmacytoma cells and spleen cells from a rat immunized with recombinant glutathione-S-transferase (GST)-BimL fusion protein. Secondary staining with fluorochrome-labeled anti-rat Ig antibodies allowed detection of clones expressing Bim-specific antibodies. The screening procedure was rapid and efficient, and most monoclonal antibodies identified were proven to be useful for immunofluorescence staining and other applications.