PyEDA is a comprehensive software tool designed for the analysis of protein dynamics through Essential Dynamics (ED) and Normal Mode Analysis (NMA) of NMR and Molecular Dynamics (MD) structures. The software is capable of processing PDB files, superimposing structures, and analyzing eigenvectors to quantify protein motions. In this study, PyEDA’s performance was validated by reproducing key results from Van Aalten et al. (Methods 14:318–28, 2011) on the NMR structure of the Mouse c-Myb R2R3 DNA-binding domain. The software successfully identified hinge-bending motions between R2 and R3 repeats, consistent with the original findings. Additionally, PyEDA was tested with the Envz protein from Escherichia coli and MD data from a hen egg-white lysozyme, demonstrating its versatility across different protein structures and data types. Despite some limitations, including a dependency on multiple Python packages and an unresolved bug with the “END” keyword on macOS, PyEDA proves to be a valuable and user-friendly tool for bioinformaticians. Future developments aim to integrate PyMOL into the GUI for enhanced visualization capabilities.