Vancomycin is not appreciably passaged via the colonic membrane to the gastrointestinal (GI) tract in persons with an intact gut epithelium due to its large chemical structure. However; hospitalized patients with diarrhea often have a disrupted GI tract. The aim of this study was to determine the frequency of detectable vancomycin concentrations in the stool of patients with antibiotic-associated diarrhea receiving IV vancomycin. This was a multicenter cohort study of hospitalized patients with stool samples collected for Clostridioides difficile testing. Leftover stool samples were collected from patients who had received at least 3 days of IV vancomycin. Fecal vancomycin was quantified by high-performance liquid chromatography. The study cohort included 33 unique patients, majority female (54.5%) aged 60 years (range 23–84). Eighteen of thirty-three patients (54.5%) tested positive for C. difficile toxins. The average duration of systemic vancomycin administration prior to stool collection was 3.5 (range 2–15) days. Three of 33 (9%) stool samples had a detectable vancomycin concentration (range 1.2–13.2 mcg/mL). These concentrations may promote the development of vancomycin-resistant Enterococcus or van mutations in C. difficile, leading to vancomycin resistance. Further studies on implications are warranted.