Updated project metadata. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful protein footprinting technique to study protein dynamics and binding; however, HDX-MS data analysis is often challenging and time consuming. Moreover, the HDX community is expanding to investigate multi-protein and highly complex protein systems such as cell lysates which further complicates data analysis. Thus, a simple and easy-to-use open source software package designed to analyze large and highly complex protein systems is needed. In this vein, we have developed The Deuterium Calculator, a Python-based software package for HDX-MS data analysis. The Deuterium Calculator is capable of differential and nondifferential HDX-MS analysis, produces standardized data files according to the recommendations put forth by the International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX) to increase transparency in data analysis, and generates Woods’ plots for statistical analysis and data visualization. This standard output can be used to perform further analysis on HDX such as determination of labeling time dependent deuteration and for the study of protein folding kinetics or differential uptake. Moreover, the Deuterium Calculator is capable of analyzing large HDX-MS datasets (e.g., LC-HDX-MS from complex samples such as cell lysates) to determine the extent of deuteration on individual peptides from numerous proteins, perform differential analysis under varying experimental conditions, and time-dependent deuterium exchange. The Deuterium Calculator is freely available for download at https://github.com/OUWuLab/TheDeuteriumCalculator.git.