GSATools is a free software package to analyze conformational ensembles and to detect functional motions in proteins by means of a structural alphabet. The software integrates with the widely used GROMACS simulation package and can generate a range of graphical outputs. Three applications can be supported: (i) investigation of the conformational variability of local structures, (ii) detection of allosteric communication, and (iii) identification of local regions that are critical for global functional motions. These analyses provide insights into the dynamics of proteins and allow for targeted design of functional mutants in theoretical and experimental studies.