C' dots are fluorescent inorganic-organic hybrid nanoparticles synthesized in water comprised of a silica core with a covalently embedded near infrared dye, and a polyethylene glycol (PEG) outer layer. C' dots containing the integrin specific ligand, cycloRGDyC, are the first of their kind particles approved for human clinical trials. In the continued clinical development of these nanoparticles, high-resolution analytical approaches are needed. Here we investigate the use of reversed phase high performance liquid chromatography (RP-HPLC) to analyze cycloRGDyC-Cy5-C' dots. Given the stability and protein-like size, we reasoned that these nanoparticles would be compatible under RP-HPLC conditions typically used to characterize peptides and proteins. Our results show that RP-HPLC provides excellent resolution, showing significant heterogeneity of these nanoparticles. C' dots also exhibit unusual peak profiles where RP-HPLC chromatogram peak shapes change from run to run, possibly due to the conformational heterogeneity or charge distribution of the particle surface due to the PEG groups. In addition we describe a novel thiol-mediated release of C' dot ligands to directly estimate cycloRGDyC by exposing the particles to organic thiols. Ligand release is presumably afforded by a reverse Michael reaction mechanism.