Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality worldwide, and early diagnosis needs to be improved. We examined whether neutral desorption extractive electrospray ionization mass spectrometry (ND-EESI-MS) could be used to detect sputum lipids expression changes to enable earlier diagnosis. Overall, 167 NSCLC patients and 140 controls were enrolled. The main peaks in the sputum of patients with NSCLC patients differed from controls (83.3% of total variability), and the signals were not associated with pathological type, TNM stage or smoking history. The relative abundance of peaks at m/z734, m/ z756, m/z772, m/z782, m/z798 and m/z803 reliably distinguished NSCLC sputum from control. Collision-induced dissociation confirmed that m/z734, m/z756, and m/ z772 represented [DPPC + H]+, [DPPC + Na]+, and [DPPC + K]+, respectively, and m/ z782, m/z798, and m/z803 represented sphingomyelin, phosphatidylglycerol, and phosphatidylglycerolphosphate, respectively. The relative abundance of DPPC was clearly lower in NSCLC sputum than in control, and the relative abundances of phosphatidylglycerol and phosphatidylglycerolphosphate were higher in NSCLC sputum than in control. The detection of changes in sputum lipids with ND-EESI-MS may be a noninvasive, radiation-free, relatively inexpensive, repeatable, and efficient method for diagnosis of NSCLC.