In this study, we successfully produced a scaffold-free cell construct for regenerative medicine using the Kenzan method, which involves the fusion of spheroids on a needle array. The cardiac construct fabricated using a three-dimensional bioprinter reproduced heart beating on the needle array, and the resultant displacement of the micro-Kenzan needle was proposed as a non-invasive procedure to analyze the cardiac constructs. Therefore, this study aimed to develop a software that accomplishes this non-invasive analysis by applying image processing technology, using a video of the cardiac construct taken directly overhead the construct. The Lucas–Kanade method was used to calculate the optical flow for tracking the needle tips. The developed software was used to measure the displacement of the needle tip over time, and the results showed that the software was able to evaluate the waveforms and beating direction of the entire or partial cardiac construct.