Background: Guthrie implemented dried blood spot (DBS)-based bioanalytical techniques in the 1960s in order to screen for phenylketonuria (PKU) disease in neonates. Historically, the dominant matrix analyzed using the “Guthrie card” platform has been whole blood, but recent advances in device technology now allow for whole blood-to-plasma sample processing prior to drying and subsequent bioanalysis. This project aimed to use this dried matrix technologies in an entirely new direction by performing dried fecal spot (DFS)-based bioanalysis for analytical microbiology and microbiome applications. Homogenized fecal sample extracts were loaded onto quantitative DBS (qDBS) devices and were allowed to dry overnight, then were shipped at ambient shipping temperature. The DBS spots were processed and subjected to LC-MS/MS-based targeted metabolomics using our Glutamate Cycle method (Glu, Gln, and GABA). Methods & Results: Fecal samples were collected from each of the five human subjects enlisted in the following cohorts: 1) healthy controls; 2) patients with diarrhea; and, 3) Clostridioides diffcile-infected (CDI) patients. A volume of ice-cold methanol:water (1:1, v:v) equivalent to a fecal density of 50 mg/mL was added into individual homogenization tubes that each contained 100 mg of 1.4 mm silica beads. Fecal samples were homogenized using 5 bead-beating cycles (4 m/s for each 20 s pulse). After centrifugation (@500 g), a 20-μL sample supernatant volume was loaded onto qDBS devices that yielded an exact 10-μL DFS spot volume on the embedded cards. Dried DFS spots were removed from the device and homogenized in a 200-μL volume of an IS solution. The Glu, Gln, and GABA concentration of each sample was compared against the concentrations measured in the matched frozen fecal extracts. We were able to successfully detect GABA in both the standard extraction method and in our processed DFS samples. Interestingly, we observed large variability between patient samples and there were no statistically significant differences between the cohorts. Conclusion: Collectively, these data demonstrate the utility of our method for measuring the concentrations of Glu, Gln and GABA contained in our processed DFS-based sample sets. This study was supported by the NIH K01K123195-01 (MAE). The Texas Children’s Hospital Department of Pathology provides salary support to Texas Children’s Microbiome Center-Metabolomics Lab staff, and purchased all of the reagents, the consumables and durable supplies, and the liquid chromatography-tandem mass spectrometry (LC-MS/MS) equipment described. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.