Abstract The structures of lipids are diverse, and thus, lipids show various biological functions. Systematic determination of lipids in organisms has always been a concern. In this paper, a methodology on the matrix-assisted laser desorption ionization mass spectrometry (MALDI MS), with titanium dioxide nanoparticles (TiO₂ NPs) as the matrix, was studied for lipid determination. The results showed that the following conditions were preferable in the determination of small-molecule lipids (such as hypoxanthine, guanosine, uridine, and cytidine), lipid standards (such as GC, GM, TG, phosphatidylethanolamine, phosphatidylcholine, and ceramide), and mixed lipids (extracted from brain homogenate with methanol alone and with the B&D method): TiO₂ NPs as the matrix, absolute ethanol as the solvent, 1 mg of TiO₂ NPs dispersed in 1 mL of absolute ethanol as the matrix solution, NaCl as the ionization reagent, and positive mass spectrometry (MS) as the mode. Modified TiO₂ NP as a new matrix for MALDI MS will be a future research direction; in addition, the characteristics of TiO₂ NPs make it a potential matrix for imaging MS.