Abstract: Objective: To establish a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the detection of serum oleic acid (OA), and preliminarily evaluate the role of OA in insulin resistance (IR) of type 2 diabetes (T2DM). Methods: OA-［¹³C₅］ was used as isotope-labeled internal standard, and the ion pairs of OA and OA-［¹³C₅］ were 281.3/281.3 and 286.3/286.3, respectively. The ultrapure water was used as mobile phase A and methanol: acetonitrile (1∶1, v/v) as mobile phase B in a ZORBAX SB-Aq C18 reversed phase column. Meanwhile, the gradient elution system with a flow rate of 0.3 mL/min was used. According to the CLSI guidelines (EP15-A3), the reliability of the established method was evaluated by detecting the performance indicators such as precision, trueness, linear range, stability and carrying contamination rate. Serum OA levels were detected by the established HPLC-MS/MS method in 109 patients with clinically diagnosed T2DM and 100 healthy controls. The insulin resistance index (HOMA-IR) was calculated to evaluate IR, and the relationship between OA and IR was further analyzed. Results: The established HPLC-MS/MS method for the detection of serum OA had good specificity and linearity in the range of 10-1 000 μmol/L (y=0.007 55x+0.004 83,r=0.997 7), and the low limit of quantification (LLOQ) was 10 μmol/L. It also had good precision, and the within-run coefficient of variation (CV) and total CV were not more than 1.62% and 1.73%, respectively, indicating that the method was suitable for the detection of serum OA. The serum OA levels in T2DM patients ［(425.58 ± 220.17) μmol/L］ were significantly higher than that in the healthy controls ［(113.20±58.00) μmol/L］, and serum OA levels were significantly correlated with HOMA-IR in T2DM patients and healthy controls. The area under the receiver operating characteristic (ROC) curve (AUC) of OA for the diagnosis of IR was 0.689. When the cut-off value identified by Youden index was 235.8 μmol/L, the sensitivity and specificity were 70.4% and 63%, respectively. When OA combined with fasting blood glucose (FBG) to diagnose IR, the AUC increased to 0.806, which was significantly higher than that of OA (P＜0.05). Conclusion: A scientific and efficient HPLC-MS/MS method for the quantitative detection of serum OA is established successfully, which provides a reliable method for the dynamic monitoring of the changes of OA levels in the patients with metabolic diseases.