The GM2 activator protein (GM2AP) is an essential component in the degradation pathway of neuronal gangliosides. GM2AP is a required accessory protein for the hydrolytic conversion of GM2 to GM3 by a water soluble hydrolase. The X-ray structure of GM2AP reveals a β-cup topology with multiple conformations of the protein within the unit cell. Because the crystal structures show different conformations of the putative membrane binding loops, we have utilized site-directed spin labeling to investigate conformational flexibility of these loops for protein in solution and bound with GM2 ligand. As such, a series of single and double CYS mutants (still with original 8 CYS in 4 disulfide bridges) have been generated and spin labeled with MTSL. EPR spectra of spin labeled GM2AP were collected with and without GM2 ligand, and no significant changes in the EPR lineshape were seen. EPR spectra were simulated for spin labels located in the loop regions and reveal multiple component fits, while those in the backside of the β-cup beta strands have single component fits. For certain sites in the mobile loops, spectra were acquired as a function of temperature. From these lineshape simulations, the activation energy for the conformational change has been determined. The SDSL EPR results indicate that the multiple conformations observed in the crystallographic unit cell are populated in solution and represent conformational flexibility of the protein; which is not necessarily altered by binding to lipid ligands. Additionally, spin labeled protein was analyzed by mass spectrometry to confirm proper formation of the four disulfide linkages and addition of only one spin label at the mutant cysteine.