This study aimed to assess the motion and its deformation index (DI) of red blood cells (RBCs) flowing through a microchannel with a microstenosis using an image analysis-based method. For this purpose, a microchannel having a smooth contraction was used and the images were captured by a standard high-speed microscopy system. An automatic image-processing and analysing method was developed in a MATLAB environment to not only track the motion of RBCs but also measure the DI along the microchannel. The keyhole model, tested in this study, proved to be a promising technique to automatically track individual RBCs in microchannels.