Total internal reflection fluorescence (TIRF) microscopy has permitted visualization of myosin and actin mechanochemistry on a single-molecule level. It has, for instance, been possible to study complex phenomena such as temporal relationship between ATP turnover and the force producing transition. However, despite quite large number of studies using fluorescent ATP analogs, several challenges remain in the interpretation of TIRF data in terms of ATP binding to the active site of myosin. We here present improvements in assay conditions for more reliable detection of ATP on-time (i.e. dwell time) at the active site(s) of myosin using approaches to suppress fluorophore blinking events related to the photophysics of the fluorescent probe. We also suggest assay designs for analysis of myosin-binding to actin filaments on a surface in the presence of nucleotide. The experiments were performed using a custom built TIRF microscopy platform and fluorescent Alexa Fluor 647 ATP (Alexa-ATP) whose turnover to Alexa Fluor 647 ADP (Alexa-ADP) and inorganic phosphate (Pi) by myosin and actomyosin occurs by a similar mechanism as for unlabeled ATP. Using this system we present solutions for improved single molecule assays for actin and myosin with emphasis on the use of reducing and oxidizing agents (trolox/trolox-quinone; TTQ) in assay buffers to suppress photo-blinking while maintaining long-lasting fluorescence signals. The cumulative frequency distribution of dwell-time events exhibited more than one exponential phase both in the presence and absence of TTQ. The relative amplitude of the slowest phase increased appreciably in the presence of TTQ as well as the overall half-time.