The development of miniaturized assays for the actin based molecular motors of the myosin type, is crucial to reduce the workload and time to extract important information associated with the chemomechanical cycle of the motor. It is also important with such assays to better understand a plethora of associated myopathy-causing mutations. In previous studies, single molecule methods involving TIRF microscopy have been established to study the basal myosin ATPase activity. The aim of this study was to further upgrade the single molecule assay to be able to extract the corresponding actin-activated myosin ATPase activity. Inspired by previous studies, crosslinking of human β-cardiac myosin on top of surface-immobilized F-actin was performed using the crosslinker EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride). The results suggest promising crosslinking efficiency of myosin on top of F-actin. Myosin molecules thus persisted on the filaments despite the introduction of ATP that otherwise dissociates the actomyosin complex. Three different methods of immobilizing F-actin to a nitrocellulose surface were investigated, biotin-streptavidin-, NEM-HMM-based and direct crosslinking of F-actin to nitrocellulose. Overall crosslinking of F-actin to nitrocellulose shows reliable immobilization of filaments while the other methods examined have their advantages and disadvantages. The results suggest that further optimization is required for the biotin-streptavidin and NEM-HMM based attachment. Using the developed assay, we were able to detect binding of fluorescently labelled ATP to myosin, cross-linked on top of F-actin, at single molecule resolution.