In this work, the depth profile analysis capability with direct current glow discharge mass spectrometry (dc-GD-MS) was evaluated by examining molybdenum disulfide (MoS2) films on Al and steel substrates. The optimized glow discharge conditions for obtaining an ideal flat crater and an efficient signal intensity were a discharge current of 1.0–1.5 mA and a discharge pressure of 4.7 mPa. The dc-GD-MS depth profile analysis provided depth resolutions of 0.55 μm for the MoS2/Al sample and 0.70 μm for the MoS2/Steel sample. The interface of 4.46 μm for MoS2/Al and 4.55 μm for MoS2/Steel determined by dc-GD-MS was close to the thicknesses of 4.85 μm and 5.45 μm, respectively, as measured by field emission scanning electron microscopy (FE-SEM). A high-carbon steel standard sample (NIST SRM 1264a) was used to validate the reliability and accuracy of the method. Relative errors of less than 12% were obtained compared with the certified concentration and the relative standard deviation (RSD, n = 20) of typical elements within 10%. The dc-GD-MS depth profile analysis provided an efficient and reliable approach for the depth analysis of the MoS2 film.