Necrostatin-1 (Nec-1) is known as a specific and potent inhibitor of non-apoptotic cell death. In this study, a rapid and sensitive LC–MS/MS method that was developed for the determination of Nec-1 levels in plasma. Meanwhile, it has been used to explore pharmacokinetics and bioavailability of Nec-1 among rats. The m/z 260.1→131 was selected as the optimal MRM transition in analyzing Nce-1. The chromatographic separation was performed with SB-C18 analytical column using the optimized gradient elution mode. The extraction recoveries of Nec-1 ranged from 85.40% to 98.25% and the matrix effects were between 94.73% and 99.26%. Both the intra- and inter-day precision did not exceed 10.0%, respectively. Moreover, it is found that Nec-1 remained stable in plasma despite different processing and storage environment. The plasma concentration of Nec-1 was successfully determined among rats who received single dose via intravenous and oral route (5mg/kg), respectively. A two-compartment model was fitted the concentration-time profile of the Nec-1 with C_max 1733μgL⁻¹ and t_1/2 1.8h for intravenous route, and C_max 648μgL⁻¹ and t_1/2 1.2h for oral route, respectively. The results showed that absolute bioavailability of Nec-1 was 54.8%. It is promising that the study is helpful to understand in vivo behaviors of Nec-1 and facilitate the future investigations of the compound.