A simple and sensitive high-performance liquid chromatography (HPLC) assay for the analysis of CZ48, a potent anticancer candidate, and its active metabolite camptothecin (CPT) in mouse plasma was developed and validated. CZ44 was used as an internal standard (IS). The samples were injected onto a C18 Synergi Polar-RP column (4μm, 150mm×4.60mm) maintained at 30°C. The identification of peaks showed high specificity. Shimadzu RF-10AXL fluorescence detector was used at the excitation and emission of 380 and 418nm, respectively. The mean recoveries were 81.41±0.035%, 86.00±0.053% and 82.21±0.020% for CZ48 and 76.01±0.028%, 77.04±0.042% and 85.93±0.023% for CPT at three concentrations of 10, 100 and 900ng/ml, respectively. The calibration curve was linear (r 2 =0.9999) over CZ48 and CPT concentrations ranging from 5 to 1000ng/ml and 10–1000ng/ml (n=6), respectively. The method had an accuracy of >95% and intra- and inter-day precision (RE%) of <1.2% and <2.2% for CZ48 and CPT, respectively, at three different concentrations (10, 100 and 900ng/ml). The lower limit of quantification (LLOQ) using 0.1ml mouse plasma was 10ng/ml for CZ48 and 5ng/ml for CPT. Stability studies showed that CZ48 and CPT were stable in mouse plasma after 4h incubation at room temperature or after 1 month storage at −80°C with three freeze/thaw cycles. The method reported is simple, reliable, precise and accurate and confirmed by the determination of plasma samples in the mice after oral administration of CZ48.