Mass spectrometry is a powerful tool in the investigation of the human fecal metabolome. However, current approaches require time-consuming sample preparation, chromatographic separations, and consequently long analytical run times. Rapid evaporative ionization mass spectrometry (REIMS) is a method of ambient ionization mass spectrometry and has been utilized in the metabolic profiling of a diverse range of biological materials, including human tissue, cell culture lines, and microorganisms. Here, we describe the use of an automated, high-throughput REIMS robotic platform for direct analysis of human feces. Through the analysis of fecal samples from five healthy male participants, REIMS analytical parameters were optimized and used to assess the chemical information obtainable using REIMS. Within the fecal samples analyzed, bile acids, including primary, secondary, and conjugate species, were identified, and phospholipids of possible bacterial origin were detected. In addition, the effect of storage conditions and consecutive freeze/thaw cycles was determined. Within the REIMS mass spectra, the lower molecular weight metabolites, such as fatty acids, were shown to be significantly affected by storage conditions for prolonged periods at temperatures above −80 °C and consecutive freeze/thaw cycles. However, the complex lipid region was shown to be unaffected by these conditions. A further cohort of 50 fecal samples, collected from patients undergoing bariatric surgery, were analyzed using the optimized REIMS parameters and the complex lipid region mass spectra used for multivariate modeling. This analysis showed a predicted separation between pre- and post-surgery specimens, suggesting that REIMS analysis can detect biological differences, such as microbiome-level differences, which have traditionally been reliant upon methods utilizing extensive sample preparations and chromatographic separations and/or DNA sequencing.