Photocyanine, a novel amphoteric phthalocyanine drug, showed favorable anticancer activity in vivo. Pharmacokinetic study in cancer patients is an important component in dose administration choice. In this study, a rapid, sensitive analytical method based on high-performance liquid chromatography with ultraviolet detection was developed and validated for the determination of four isomers of photocyanine (FD1-4) in cancer patients. Sample preparation involved liquid-liquid extraction with a combination of ultrasound and N,N-dimethyl formamide. Calibration curves (1/x(2)) offered satisfactory linearity for the four isomers of photocyanine. The lower limit of quantification (LLOQ) for FD1-3 isomers was 30 ng/mL, and LLOQ for FD-4 was 5 ng/mL. Inter- and intra-day accuracies for four isomers ranged from 96.6 to 105.5%, and 95.0 to 103.6%, respectively. Inter- and intra-day precision ranged from 4.8 to 8.9%, and 3.6 to 12.2%, respectively. Stability studies showed that photocyanine was stable. This method was successfully used to quantify photocyanine in a pharmacokinetic study in which a single-dose of photocyanine (0.1 mg/kg) was intravenously administered to patients with cancer. On the basis of the discovery that photocyanine has a half-life of 57.5 h in vivo, we suggest that avoiding light for a longer period is essential for patients undergoing photocyanine therapy.