The publication describes how to use and analyse data generated with the GTPase assay 'GTPase-Glo assay' by Promega. The data set exemplifies the protocol steps (input, analysis code, output files) described in the accompanying publication, using 2 concrete examples. The data set contains the following files: (1) assay data (luminescence/ amount of remaining GTP for various time points and GTPase enzyme concentrations) for Cdc42 and Ras. (2) a python scirpt used to reformat the data. (3) matlab code for data analysis. (4) output data (rates and plots) that the matlab code generates.