Laser scanning microscopy has proven to be a transformative tool in the biomedical sciences. The technology provides a means to image three-dimensional, time-varying, micro-scale features within living tissue, which in turn has borne exciting avenues for the experimental investigation of the relationship between the hierarchical spatial scales within organisms. By coupling laser scanning microscopes to mechanical testing systems, one can study the relationships between mechanics at the organ, tissue, and cellular scales. In practice, while a controlled mechanical state is applied to intact tissue, images of the cells within can be obtained via microscopy;​ however, extracting the geometric information from these images and characterizing deformation has been a challenge to the field. pyCellAnalyst is an extensive software framework for such analysis of these image data. It provides an accessible interface to reconstruct cellular geometries from images, characterize deformation, and perform finite element analyses informed by the experimental observations.