The article introduces a rapid data analysis method for differential mobility cytometry (DMC), a technique used to capture and study cell adhesion processes. DMC employs an oscillating flow field to maintain cells in a glass channel or microfluidic device, allowing for the observation of cell interactions with an affinity surface. The traditional method of creating differential images manually has limited the throughput of DMC. To address this, the authors propose a new method that involves subtracting short movies from each other to create a stack of differential images, which can be easily analyzed using ImageJ software. This approach aims to make DMC more accessible and improve its throughput. The method is demonstrated through experimental procedures, including the preparation of PDMS-glass microdevices, cell loading, and video acquisition. The analysis process involves subtracting two images in the video sequence to highlight changes in cell positions, which are then used to calculate the mobility difference of target cells.The article introduces a rapid data analysis method for differential mobility cytometry (DMC), a technique used to capture and study cell adhesion processes. DMC employs an oscillating flow field to maintain cells in a glass channel or microfluidic device, allowing for the observation of cell interactions with an affinity surface. The traditional method of creating differential images manually has limited the throughput of DMC. To address this, the authors propose a new method that involves subtracting short movies from each other to create a stack of differential images, which can be easily analyzed using ImageJ software. This approach aims to make DMC more accessible and improve its throughput. The method is demonstrated through experimental procedures, including the preparation of PDMS-glass microdevices, cell loading, and video acquisition. The analysis process involves subtracting two images in the video sequence to highlight changes in cell positions, which are then used to calculate the mobility difference of target cells.