Objective To establish an HPLC method for simultaneous analysis of danshensu (DSS), protocatechuic acid (PA) and hydroxysafflor yellow A (HSYA) in plasma samples, and to study the pharmacokinetics of DSS, PA and HSYA in Danhong Injection in normal and cold-coagulation and blood-stasis model rats. Methods The cold-coagulation and blood-stasis rat models (n=6) were made by continuous stimulation with ice water for 20 days; another 6 normal rats served as controls. The concentrations of DSS, PA and HSYA in the plasma were determined by RP-HPLC (0.2% formic acid water[A]-methanol[B], gradient elution, wavelength detection: 280 nm [0-40 min] and 402 nm [40-60 min] ) at 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, and 90 min after administration of Danhong Injection via the tail vein. The pharmacokinetic parameters were calculated with DAS 3.0 software. Results DSS and PA had an open two compartment model and HSYA had an open three compartment model. Compared with the normal groups, the model group had significantly increased maximum plasma concentration (C_max) of DSS, PA and HSYA, the distribution half-life time (t_1/2α) of DSS, and elimination half-life time (t_1/2α, t_1/2γ), area under curve (AUC), and apparent volume (V) of PA and HSYA (P <0.05); it also had significantly reduced distribution half-life time (t_1/2α) of PA and HSYA and area under curve (AUC) of DSS(P <0.05). Conclusion DSS has a lower distribution and bioavailability under the condition of cold-coagulation and blood-stasis, while PA and HSYA show a faster distribution, slower elimination, increased apparent volume and bioavailability, indicating a better clinical effect.