Well characterized test materials are essential for validating the performance of current trace explosive detection systems. Explosive encapsulated microspheres have proven to be a valuable test material for trace explosive detection because of their precise size, shape, and composition. Presented herein is the quantification of explosives in the polymer microspheres by high performance liquid chromatography with UV/Vis detection (HPLC-UV/Vis). A size exclusion separation is employed to quantify the amount of explosive encapsulated in cured microspheres. Complete quantification was achieved by simultaneously separating and quantifying the explosive and polymer components. Results indicate that approximately 30% of the TNT is lost in the manufacturing of the microspheres and subsequent loss from the cured microspheres is minimal if stored at 4 degrees C.