We have compared the strikingly different decomposition pathways for camptothecin-20(S)-acetate -acetate and camptothecin-20(S)-glycinate in phosphate buffered saline, human plasma and blood. The aliphatic ester analog camptothecin-20(S)-acetate demonstrated excellent stability in the above fluids for many hours with minimal hydrolysis, while the camptothecin-20(S)-glycinate analog (differing solely by the presence of an amino group) underwent rapid and essentially complete decomposition. Reversed-phase high performance liquid chromatography (RP-HPLC) with electrospray ionization-mass spectral (ESI-MS) detection was then used to correlate structural information for camptothecin-20(S)-glycinate decomposition products. ESI-MS detection indicated the ring-opened carboxylate form of camptothecin and the ring-opened degradation product co-elute near the solvent front, while the latest eluting decomposition product was the closed-ring lactone form of camptothecin. A novel decomposition product with intermediate retention time displayed an identical mass-to-charge ratio as camptothecin-20(S)-glycinate ester but a strikingly different fragmentation pattern. The LC-ESI-MS evidence of a novel camptothecin prodrug degradation pathway is provided in this report.