Here, we document the discovery of a monoclonal antibody that selectively binds to both human and murine fibroblast activation protein alpha (FAP), a serine protease that is overexpressed on cancer-associated fibroblasts (CAFs), making it an attractive therapeutic target for the aiding and abetting tumor microenvironment. The lead antibody, B12, was identified from a nai ̈ve murine single-chain variable fragment antibody phage display library screened against recombinant human FAP on magnetic beads. The heavy and light chains of B12 were cloned into full-length human immunoglobulin 1 (IgG) vectors and expressed as a chimeric monoclonal antibody (B12 IgG). We engineered a drugresistant prostate cancer cell line, CWR-R1-EnzR, to express human FAP for antibody characterization and validation (R1-EnzR FAP ). B12 IgG selectively bound to the R1-EnzR FAP cells by flow cytometry and was internalized in vitro by confocal microscopy. B12 IgG was further evaluated as a near-infrared (NIR) optical imaging probe in R1-EnzR FAP and parental xenograft models. High tumor uptake and retention of the NIR probe was observed in the R1-EnzR FAP xenografts, and endogenous expression of murine stromal origin FAP was detected in the parental xenografts. Ex vivo evaluation of these models by immunohistochemistry documented B12 IgG localization to both human and murine FAP-expressing cells.