We report, for the first time, the clinical feasibility of a novel residual gas analyzer mass spectrometry (RGA-MS) method for accurate evaluation of the 13C-glucose breath test (13C-GBT) in the diagnosis of pre-diabetes (PD) and type 2 diabetes mellitus (T2D). In T2D or PD, glucose uptake is impaired and results in blunted isotope enriched 13CO2 production in exhaled breath samples. Using the Receiver operating characteristics (ROC) curve analysis, an optimal diagnostic cut-off point of the 13CO2/12CO2 isotope ratios expressed as the delta-over-baseline (DOB) value, was determined to be δDOB13C‰ = 28.81‰ for screening individuals with non-diabetes controls (NDC) and pre-diabetes (PD), corresponding to a sensitivity of 100% and specificity of 94.4%. We also determined another optimal diagnostic cut-off point of δDOB13C‰ = 19.88‰ between individuals with PD and T2D, which exhibited 100% sensitivity and 95.5% specificity. Our RGA-MS methodology for the 13C-GBT also manifested a typical diagnostic positive and negative predictive value of 96% and 100%, respectively. The diagnostic accuracy, precision and validity of the results were also confirmed by high-resolution optical cavity enhanced integrated cavity output spectroscopy (ICOS) measurements. The δDOB13C‰ values measured with RGA-MS method, correlated favourably (R2 = 0.979) with those determined by the laser based ICOS method. Moreover, we observed that the effects of endogenous CO2 production related to basal metabolic rates in individuals were statistically insignificant (p = 0.37 and 0.73) on the diagnostic accuracy. Our findings suggest that the RGA-MS is a valid and sufficiently robust method for the 13C-GBT which may serve as an alternative non-invasive point-of-care diagnostic tool for routine clinical practices as well as for large-scale diabetes screening purposes in real-time.