Molybdenum disulfide (MoS2), with its active edge sites, is a proposed alternative to platinum for catalyzing the hydrogen evolution reaction (HER). Recently, the inert basal plane of MoS2 was successfully activated and optimized with excellent intrinsic HER activity by creating and further straining sulfur (S) vacancies. Nevertheless, little is known about the HER kinetics of those S vacancies and the additional effects from elastic tensile strain. Herein, scanning electrochemical microscopy was used to determine the HER kinetic data for both unstrained S vacancies (formal potential Ev0 = −0.53 VAg/AgCl, electron-transfer coefficient αv = 0.4, electron-transfer rate constant kv0 = 2.3 × 10(–4) cm/s) and strained S vacancies (Esv0= −0.53 VAg/AgCl, αsv = 0.4, ksv0 = 1.0 × 10(–3) cm/s) on the basal plane of MoS2 monolayers, and the strained S vacancy has an electron-transfer rate 4 times higher than that of the unstrained S vacancy. This study provides a general platform for measuring the kinetics of two-dimensional material-based catalysts.