Abstract <p>Tunable diode laser absorption spectroscopy methods are applied to study gas biomarkers of gut microbiota activity. For this purpose, a hardware and software complex (HSC DLS) and a technique for multicomponent measurements of gas biomarkers in exhaled air are developed. The complex consists of a high-resolution multichannel tunable diode laser spectrometer based on four single-mode near-IR diode lasers with a fiber optic output for measuring O₂, ¹²CO₂, ¹³CO₂, CH₄, NH₃, H₂S, and H₂O gas contents, a Herriott multipass cell with a total optical length of 35 m, and a semiconductor selective sensor for background measurements of hydrogen concentrations. To find the concentration, the cross-correlation technique for processing spectra using generalized linear/nonlinear regression is employed. Concentrations of exhaled and gut gases have been measured both in experiments on laboratory animals and in clinical studies on 25 patients. For this purpose, hydrogen–methane and lactulose loading tests have been used. Highly accurate measurements of exhaled and gut gas concentrations performed on the HSC DLS allow continuous online measurement of the composition of gut microbiota biomarkers, assessment of the enzymatic activity and taxonomic composition of the microbiota, and non-invasive diagnosis of various gastro-intestinal diseases.</p>