Since movement is an important characteristic of micro-motile cells, the development of microscopic movement tracking techniques is in urgent demand. To meet this need, applications of open source software Image-J in microscopic movement tracking were illustrated with examples. Movement track reconstruction was performed in both circular and linear movement patterns. The algorithms of real time radius, VCL (curvilinear velocity), VAP (average path velocity), VSL (straight line velocity), and other descriptive parameters using original coordinate values were developed and/or explained. Microscopic movement tracking can now be conducted at low cost using Image-J. Moreover, the algorithms can be easily controlled to fit users’ study purpose.