In tracking analysis, the movement of cargos by motor proteins in axons is often represented by a time-space plot termed a ‘kymograph’. Manual creation of kymographs is time-consuming and complicated for cell biologists. Therefore, we developed KYMOMAKER, a simple system that automatically creates a kymograph from a movie without generating multiple time-dissected movie stacks. In addition, KYMOMAKER can automatically extract faint vesicle traces, and can thereby effectively analyze cargos expressed at low levels in axons. A filter can be applied to remove traces of non-physiological movements and to extract meaningful traces of anterograde or retrograde cargo transport. For example, only cargos that move at a speed of >0.4 µm/second for a distance of >1 µm can be included. Another function of KYMOMAKER is to create a color kymograph in which the color of the trace varies according to the position of the fluorescent particle in the axis perpendicular to the long axis of the axon. Such positional information is completely lost in conventional kymographs. KYMOMAKER is an open access program that can be easily used to analyze vesicle transport in axons by cell biologists who do not have specific knowledge of bioimage informatics.