Programs that aim to control vector-borne zoonotic diseases require information on zoonotic hosts and on the feeding behavior of bridging vectors that are capable of transmitting pathogens from those hosts to humans. Here we describe an assay developed to identify bloodmeals in field-collected cat fleas (Ctenocephalides felis Bouché) to assess this species' potential role as a Yersinia pestis bridging vector in a plague-endemic region of Uganda. Our assay uses a single primer set and SYBR Green I-based real-time polymerase chain reaction to amplify a segment of the 12S mitochondrial ribosomal RNA gene for identification by sequencing. The assay capitalizes on the sensitivity of real-time polymerase chain reaction and the specificity of sequencing and can be used to differentiate vertebrate bloodmeals to the genus or species level without a priori knowledge of the host community. Because real-time assays that detect vertebrate DNA are highly sensitive to human DNA contamination, we analyzed detection in artificially fed and unfed fleas to establish a Ct cutoff that optimized specificity without completely sacrificing sensitivity. Using the established cutoff, our assay detected human, rat, and goat DNA in artificially fed C. felis up to 72 h postfeeding.