Characterizing protein flexibility is an important goal for understanding the physical–chemical principles governing biological function. This paper presents a Fragment Ensemble Method to capture the mobility of a protein fragment such as a missing loop and its extension into a Protein Ensemble Method to characterize the mobility of an entire protein at equilibrium. The underlying approach in both methods is to combine a geometric exploration of conformational space with a statistical mechanics formulation to generate an ensemble of physical conformations on which thermodynamic quantities can be measured as ensemble averages. The Fragment Ensemble Method is validated by applying it to characterize loop mobility in both instances of strongly stable and disordered loop fragments. In each instance, fluctuations measured over generated ensembles are consistent with data from experiment and simulation. The Protein Ensemble Method captures the mobility of an entire protein by generating and combining ensembles of conformations for consecutive overlapping fragments defined over the protein sequence. This method is validated by applying it to characterize flexibility in ubiquitin and protein G. Thermodynamic quantities measured over the ensembles generated for both proteins are fully consistent with available experimental data. On these proteins, the method recovers nontrivial data such as order parameters, residual dipolar couplings, and scalar couplings. Results presented in this work suggest that the proposed methods can provide insight into the interplay between protein flexibility and function. Proteins 2006. © 2006 Wiley-Liss, Inc.