Muscle fiber shortening is obtained by actin filaments sliding over the myosin filaments. Our theory of the sarcomere contraction suggests that XB dynamics is determined by two distinct, slow and fast, kinetics, and the myosin head performs several stroke steps over the actin filament per hydrolysis of a single ATP molecule. Tracking the isolated single actin filament trajectory over scattered isolated myosin heads provides insight into the mechanisms that determines the actin-myosin cross-bridge (XB) dynamics. The actin filament is labeled by fluorescent dye and the data is acquired by utilizing photomultiplier. The study developed the image analysis algorithms for quantifying the dynamics of actin propulsion in these motility assays studies. The image analysis software was developed using Matlab, and included the following steps: object detection, object tracking, backbone axis identification, measurement of actin filament length, path recognition, and calculations of the local velocity at various points along the filaments. The image sets examined in this study contained 7 actin filaments. The mean velocity for the actin filaments was 14.86 ± 3.5mm/s. This velocity is significantly faster than expected velocity based on the rate of ATP. The filament lengths were constant, within the available spatial resolution. The results establish the hypothesis that the myosin head performs several stroke steps per hydrolysis of a single ATP molecule, and supports the two-kinetic theory of muscle contraction. The algorithm can be used for exploring the actin-myosin dynamics from various specimens, and for providing diagnostic test for differentiating between various cardiac diseases (cardiomyopathies) based on the actin-myosin phenotype.