Digital Holographic Microscopy (DHM) has emerged as a powerful imaging tool due to its ability to provide quantitative sample morphological data in a label-free manner. Off-axis holography further opens possibilities of numerical approaches to image analysis and data processing. However, this involves computationally heavy workflows that limit the tool’s usability in biomedical research. In this paper, a software package is developed for off-axis hologram reconstruction and processing. The software can perform a variety of bioimaging operations including imaging, region of interest selection, automated reconstruction, and data extraction, with a user-friendly graphical user interface. Originally programmed in MATLAB, the software can be installed to use in common lab-based Microsoft Windows computers. The software can be easily adopted to work with customized off-axis DHM setups, aiming to increase the efficiency of DHM’s bioimaging workflow in the community. To demonstrate this software, blood platelet experiments were conducted to show quantitative volume change of thrombus formation.