<p>Enhancing stomatal aperture to boost CO₂ uptake is a promising strategy for improving plant photosynthetic efficiency and, ultimately, biomass. Research on stomatal aperture regulation has focused primarily on Arabidopsis, crops, and vegetables, while studies on grass species remain relatively scarce. This study utilized augustinegrass (Stenotaphrum <i>secundatum</i>)—renowned for its excellent properties and wide application in greening and ecological restoration—as its experimental material. We first established a convenient method for observing stomatal aperture by incubating the adaxial leaf surface in an opening solution (0.5% KCl, pH 6.0) under a photosynthetic photon flux density of 352.2 µmol m⁻² s⁻¹ for one hour, followed by the application of nail polish to capture impressions of open stomata. This method was then used to screen for compounds that promote stomatal opening. We found that the stomatal aperture exhibited a concentration-dependent response to sixteen exogenous substances. The following compounds were identified as effective agents for increasing the stomatal aperture of augustinegrass leaves: 10 µM brassinolide; 1, 10, and 100 µM salicylic acid; 1000 nM indoleacetic acid; 0.1, 1, and 10 mM methionine; 1 and 10 mM proline; 1 and 10 mM alanine; 0.1 and 10 mM glutamate; 1 mM histidine; and 0.1 and 10 mM arginine. This study establishes a basis for developing stomatal opening promoters with the future potential to enhance photosynthesis—via increased stomatal aperture—and ultimately improve biomass production.</p>