L-amino acids are well characterized as the building blocks of proteins, but the biological roles of D-amino acids (DAA) are far less understood. D-Alanine (D-Ala) can be accumulated to substantial concentrations in the insulin-secreting beta-cells, and D-Ala release from rat islets of Langerhans upon glucose stimulation is also observed. Unlike D-Ser and D-Asp, which can be endogenously synthesized in the central nervous system of animals, D-Ala in animals is believed to be obtained from exogenous sources such as food and intestinal microorganisms. Moreover, the genes involved in D-Ala biosynthesis and metabolism are significantly altered in the gut microbiome of type II diabetes patient cohorts, suggesting important physiological roles of microbiota-derived D-Ala in regulating glucose homeostasis. To better understand underlying mechanisms, we sought to isolate and engineer individual microorganisms that secrete high levels of D-Ala from the rat gut microbiota. Serial dilutions of colon contents from adult Sprague–Dawley rats were plated on agar media to obtain clonal cultures, which were subjected to rapid strain typing using matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS). Followed by 16S ribosomal RNA sequencing analysis, forty-three unique strain isolates were confirmed. Then, a two-tier screening workflow was devised to profile D-Ala production. First, a 96-well microtiter assay using a broad-spectrum D-amino acid oxidase (DAAO) coupled with hydrogen peroxide detection was established for rapid assessment of general DAA production. Then, chiral LC-MS was utilized to quantify the levels of D-Ser, D-Asp, D-Pro and D-Ala in a more specific but time-consuming manner. To isolate spontaneous mutant strains of top producers with reduced or abolished secretion of D-Ala, we are currently incubating target microorganisms on agar media supplemented with DAAO, as reduced D-Ala production is predicted to result in less hydrogen peroxide production and therefore faster growth to facilitate mutant selection. In summary, we demonstrated the use of high-throughput analytical methodology to accelerate understanding and engineering of complex host-microbiota interactions at the molecular level. We envision engineered microbiota strains with specific modifications of biosynthetic capabilities, such as DAA production, may serve as valuable research tools to reveal how the microbiota modulates host physiology.