(S)-4'-nitro-Blebbistatin is a more stable and less phototoxic form of (–)-blebbistatin , which is a selective cell-permeable inhibitor of non-muscle myosin II ATPases. (–)-Blebbistatin rapidly and reversibly inhibits Mg-ATPase activity and in vitro motility of non-muscle myosin IIA and IIB for several species (ICs = 0.5-5 µM), while poorly inhibiting smooth muscle myosin (IC = 80 µM). Through these effects, it blocks apoptosis-related bleb formation, directed cell migration, and cytokinesis in vertebrate cells. However, prolonged exposure to blue light (450-490 nm) results in degradation of blebbistatin to an inactive product via cytotoxic intermediates, which may be problematic for its use in fluorescent live cell imaging applications. The addition of a 4’-nitro group decreases the inherent fluorescence, stabilizes the molecule to circumvent its degradation by prolonged blue light exposure, and decreases its phototoxicity while retaining the in vitro and in vivo activity of (–)-blebbistatin. (S)-4'-nitro-Blebbistatin has the same stereochemistry as the active (–)-blebbistatin enantiomer.