Quantitative and qualitative running gait analysis allows the early identification and the longitudinal monitoring of gait abnormalities linked to running-related injuries. A promising calibration- and marker-less video sensor-based technology (i.e., Graal), recently validated for walking gait, may also offer a time- and cost-efficient alternative to the gold-standard methods for running. This study aim was to ascertain the validity of an improved version of Graal for quantitative and qualitative analysis of running. In 33 healthy recreational runners (mean age 41 years), treadmill running at self-selected submaximal speed was simultaneously evaluated by a validated photosensor system (i.e., Optogait—the reference methodology) and by the video analysis of a posterior 30-fps video of the runner through the optimized version of Graal. Graal is video analysis software that provides a spectral analysis of the brightness over time for each pixel of the video, in order to identify its frequency contents. The two main frequencies of variation of the pixel’s brightness (i.e., F1 and F2) correspond to the two most important frequencies of gait (i.e., stride frequency and cadence). The Optogait system recorded step length, cadence, and its variability (vCAD, a traditional index of gait quality). Graal provided a direct measurement of F2 (reflecting cadence), an indirect measure of step length, and two indexes of global gait quality (harmony and synchrony index). The correspondence between quantitative indexes (Cadence vs. F2 and step length vs. Graal step length) was tested via paired t-test, correlations, and Bland–Altman plots. The relationship between qualitative indexes (vCAD vs. Harmony and Synchrony Index) was investigated by correlation analysis. Cadence and step length were, respectively, not significantly different from and highly correlated with F2 (1.41 Hz ± 0.09 Hz vs. 1.42 Hz ± 0.08 Hz, p = 0.25, r2 = 0.81) and Graal step length (104.70 cm ± 013.27 cm vs. 107.56 cm ± 13.67 cm, p = 0.55, r2 = 0.98). Bland–Altman tests confirmed a non-significant bias and small imprecision between methods for both parameters. The vCAD was 1.84% ± 0.66%, and it was significantly correlated with neither the Harmony nor the Synchrony Index (0.21 ± 0.03, p = 0.92, r2 = 0.00038; 0.21 ± 0.96, p = 0.87, r2 = 0.00122). These findings confirm the validity of the optimized version of Graal for the measurement of quantitative indexes of gait. Hence, Graal constitutes an extremely time- and cost-efficient tool suitable for quantitative analysis of running. However, its validity for qualitative running gait analysis remains inconclusive and will require further evaluation in a wider range of absolute and relative running intensities in different individuals.