Increasing interest in self-assembled, protein-mimetic structures formed from peptide-amphiphile building blocks has created a need for new techniques to characterize these assemblies. Unlike typical surfactant systems, incorporation of fluorophores, such as tryptophan, facilitates simple spectroscopic characterizations. This work demonstrates the use of fluorescence anisotropy in tryptophan-containing peptide-amphiphiles to determine their critical micelle concentration (CMC). Below the CMC, the individual amphiphiles have little anisotropy, but as they aggregate, their rotational diffusion slows, increasing the amount of anisotropy in the system. This spectroscopic technique is compared to traditional methods for CMC determination such as pyrene solubilization and solution conductivity.