This document analyzes the strain hardening behavior of an Fe-22wt.% Mn-0.6wt.% C twinning-induced plasticity (TWIP) steel under tensile deformation. Electron channeling contrast imaging (ECCI) combined with electron backscatter diffraction (EBSD) is used to characterize the evolution of dislocation and twin substructures. The analysis reveals that strain hardening occurs in five stages with different rates that are correlated to the refinement of the dislocation and twin substructures. At high strains, limited further refinement of these substructures reduces strain hardening capabilities.