We present an image-matching-based automated algorithm capable of accurately determining the self-rotational speed of cancer cells in an optically-induced electrokinetics-based microfluidic chip. To automatically track a specific cell in a video featuring more than one cell, a background subtraction technique was used. To determine the rotational speeds of cells, a reference frame was automatically selected and curve fitting was performed to improve the stability and accuracy. Results show that the algorithm was able to accurately calculate the self-rotational speeds of cells up to ∼150 rpm. In addition, the algorithm could be used to determine the motion trajectories of the cells. Potential applications for the developed algorithm include the differentiation of cell morphology and characterization of cell electrical properties.