Abstract: Compared with human insulin, insulin lispro shows a faster hypoglycemic effect and a higher peak plasma concentration, which can better control postprandial hyperglycemia. In this study, we used a solid phase extraction pretreatment method and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify insulin lispro in rat plasma. Bovine insulin was used as an internal standard. Plasma samples were separated on an ACQUITY UPLC Peptide CSH C₁₈ column (2.1 mm × 50 mm, 1.7 μm) after solid phase extraction. Positive electrospray ionization was performed using multiple reaction monitoring (MRM) with transitions of m/z 1 162.5→217.2 for insulin lispro and m/z 1 157.5→136.0 for insulin bovine (internal standard). The method validation results showed that the linear range was 0.1 ng·mL^-1-100 ng·mL^-1; intra- and inter-day accuracy and precision met the acceptance criteria for biological sample analysis. The recovery of insulin lispro ranged from 63.1% to 68.1%. The method was applied in a pharmacokinetic study of insulin lispro following a single-dose subcutaneous administration to rats. Animal experiments were approved by the Experimental Animal Ethics Committee of Shanghai Institute of Materia Medica, Chinese Academy of Sciences.