Background: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been suggested to play roles in various diseases, yet there is little data on their changes in patients with non-small-cell lung cancer (NSCLC). A simple LC-MS/MS method for EPA and DHA determination is critical to exploring EPA and DHA level changes in NSCLC patients.Methods: 25 mu L of serum was mixed with 25 mu L of internal standard working solution, and then 450 mu L of acetoni-trile for protein precipitation. After vortex and centrifugation, the supernatant was directly used for LC-MS/MS analysis. The method was well validated with linearity, precision, recovery, and matrix effect. The concentrations of EPA and DHA in serum samples from 211 NSCLC patients and 227 healthy controls were determined by this LC-MS/MS method.Results: Good separation and reliable quantification of EPA and DHA in serum samples were achieved by our method. Compared with healthy controls, serum EPA and DHA were significantly reduced in both adenocarci-noma and squamous cell carcinoma patients. The concentrations of EPA and DHA showed a progressive decrease in healthy controls, early-and advanced-stage NSCLC patients.Conclusions: This study identified significant reductions in serum EPA and DHA in NSCLC patients through the development of an LC-MS/MS method.