In recent years, a great deal of attention has been paid to the combined use of multiple antitumor drugs for better cancer treatment. The aims of the study are to construct a nanoparticle drug delivery system for the co-delivery of irinotecan hydrochloride and curcumin and to develop an analytical method for simultaneously quantifying these molecules, which is essential for further studies of the co-delivered nano system. The irinotecan hydrochloride and curcumin co-delivered nanoparticle (ICN) were prepared by combinatorially entrapping them into polyethylene glycol-poly lactic acid-co-glycolic acid (PEG-PLGA) polymeric nanoparticles. A simple, sensitive and rapid high-performance liquid chromatography method was developed and validated to simultaneously quantify the compounds in the co-delivered nanoparticle system. Acetonitrile and ultrapure water containing sodium dodecyl sulfate (0.08mol/L), disodium phosphate (Na2HPO4, 0.002mol/L) and acetic acid (4%, v/v) were used as the mobile phase and their ratio was set at 50:50. The flow rate was set to 1.0mL/min, and the temperature in the column oven was maintained at 40°C. The analysis was carried out at 256 and 424nm to assess irinotecan hydrochloride and curcumin, respectively. Detectors with only one channel can also visualize both analytes in one chromatogram at 379nm and still demonstrate acceptable sensitivity. The retention times for irinotecan hydrochloride and curium were 3.317 and 5.560min, respectively. The method developed was confirmed to be sensitive, accurate (recovery, 100±2%), precise (relative standard deviation, RSD≤1%), robust and linear (R2≥0.9996) in the range from 2.05 to 1050μg/mL. The presented method has been used to quantify irinotecan hydrochloride and curcumin in the co-delivered ICN nano system to assess the drug delivery quality of the nanoparticles and can also be used for routine analysis because of its simplicity and accuracy.