A simple method for the detection of oxysterols in oxidatively modified LDL (Ox-LDL) has been developed using MALDI-TOF MS. To identify the ion peaks of oxysterols, seven major oxysterols in Ox-LDL (7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol, 5α,6α-epoxycholesterol, 5β,6β-epoxycholesterol, 25-hydroxychokesterol, (25R)-26-hydroxycholesterol), and cholesta-3,5-dien-7-one were analyzed by MALDI-TOF MS. Among these oxysterols, 7-ketocholesterol, a very abundant oxysterol in Ox-LDL, was found to show a characteristic peak of [M + H]+ at m/z 401. Cholesta-3,5-dien-7-one, which is known as a degradation product of 7-ketocholesterol upon saponification of Ox-LDL, gave a major peak of [M + H]+ at m/z 383. In contrast, other oxysterols showed similar peak patterns at m/z 367 and 385. These results were applied to the analysis of Ox-LDL by MALDI-TOF MS after saponification and hexane-extraction, detecting ion peaks at m/z 367, 383, 385, and 401. This MALDI-TOF MS method has a potential as a simple tool to show the presence of oxysterols in Ox-LDL without derivatization and chromatographic separation.