– (S)-3′-hydroxy-Blebbistatin is a more stable and less phototoxic derivative of (–)-blebbistatin, retaining the latter’s properties as a selective, cell-permeable inhibitor of non-muscle myosin II ATPases. Unlike its predecessor, (S)-3′-hydroxy-Blebbistatin exhibits reduced fluorescence, improving its utility in live cell imaging applications by diminishing degradation into cytotoxic intermediates under blue light exposure (450-490 nm). It effectively inhibits the Mg-ATPase activity and in vitro motility of non-muscle myosin IIA and IIB across various species with IC50 values ranging from 0.5-5 µM and shows minimal inhibition against smooth muscle myosin with an IC50 of 80 µM. This compound plays a critical role in preventing apoptosis-related bleb formation, directed cell migration, and cytokinesis in vertebrate cells, mirroring the active (–)-blebbistatin enantiomer in mechanism and efficacy.