Near infrared spectroscopy possess great potential for in vivo quantitative monitoring of drugs in animal subject. The accuracy of the measurements by near infrared technique should be evaluated by an established method. In this study, a near infrared fluorescence dye, cypate and its conjugation cypate-PEG were used as model drug for in vivo dynamic study. The pharmacokinetics of the model drug in mice subjects were investigated by near infrared spectroscopy and high performance liquid chromatography, respectively. The results from the two techniques were compared. The pharmacokinetic parameters calculated based on the acquired data by DAS software showed that there were no statistical differences between the two methods. The dynamic distribution of the model drugs in mouse model imaged by NIR image system indicated that cypate firstly accumulated in liver and was cleared from the enteron system, while cypate - PEG clearance from the urine system. Results indicated that NIR monitoring technique provide a promising quantitative way for in vivo monitoring the dynamics of drugs in animal subjects.