Efficient hole injection is crucial for the optimal functioning of organic light-emitting diodes (OLEDs), which require an anode system with a high work function. MoO 3 is commonly used for the hole injection layer (HIL) in OLEDs owing to its significantly high work function. However, the work function of the MoO 3 layer varies with thickness, which can affect the position of the highest occupied molecular orbital (HOMO) of the adjacent organic hole transport layer. Therefore, it is essential to understand the energy-level alignment of MoO 3 HILs with different thicknesses to design an efficient OLED structure. In this study, the energy-level alignment of indium tin oxide (ITO)/MoO 3 (20 nm)/N,N ′ -di(1-naphthyl)-N,N ′ -diphenyl-(1,1 ′ -biphenyl)-4,4 ′ -diamine (NPB) interfaces was investigated using in situ X-ray and ultraviolet photoelectron spectroscopy, and the results were compared with those of the ITO/MoO 3 (5 nm)/NPB interfaces. The 20 nm thick MoO 3 layer exhibited a high work function, leading to a significant decrease in the NPB HOMO level. These findings suggest that a sufficiently thick MoO 3 HIL is necessary to achieve optimal energy-level alignment and enhance the hole injection properties in OLEDs.