Filth flies are commonly implicated in pathogen transmission routes due to their affinity for vertebrate waste and their synanthropic associations. However, solidifying the link between flies and infected feces in the wild can be difficult, as interpretations made solely from microbial culturing or sequencing methods may represent an incomplete picture of pathogen acquisition. We present an analytical assay using high performance liquid chromatography tandem mass spectrometry (HPLC MS/MS) to detect vertebrate fecal metabolites (urobilinoids) in adult blow fly guts. Proof of concept experiments consisted of controlled feeding in which flies were grouped into three treatments (unfed, exposure to beef liver tissue, and exposure to canine feces; N = 20/treatment) using the black blow fly Phormia regina Meigen (Diptera: Calliphoridae). It was revealed that only feces-related samples exhibited peaks with an m/z of 591 and MS/MS spectra consistent with urobilinoids. These peaks were not seen for beef liver tissue, flies exposed to beef liver tissue, or unfed flies. Samples taken directly from beef liver tissue and from feces of several animals were also tested. To test this assay in wild flies, 216 flies were additionally analyzed to determine whether they had ingested vertebrate feces. About 13% of the wild flies exhibited these same peaks, providing a baseline measure of blow flies collected in urban and residential areas consuming feces from the environment. Overall, this assay can be used for P. regina collected in an applied setting and its integration with microbial culturing and sequencing methods will help to improve its use.