Graphene oxide (GO) has shown great potential as a component in various devices due to its excellent solution processability and two-dimensional structure. However, the oxygenated form of graphene has a moderate charge-transport capability. The latter parameter may be enhanced through controlled deoxygenation of GO with subsequent tuning of its work function (WF). Various reduction approaches were employed to investigate the effect of the oxygen content on the work function of GO derivatives as thin films on an indium tin oxide substrate. Such films were reduced by stepwise thermal annealing in ultrahigh vacuum up to 650 °C, by chemical reduction with hydrazine, or by a combination of chemical and thermal reduction processes. The effect of the GO film thickness and the flake size on the WF was also investigated. UV photoelectron spectroscopy and X-ray photoelectron spectroscopy were used to correlate the WF of GO derivatives with their oxygen content. The results showed that the WF is strongly dependent on the oxygen content, reaching a ∼1 eV difference between GO and highly reduced GO, under the specific reduction conditions. The film thickness affects the work function, since in thin films interaction with the substrate is pronounced. Finally, the WF of reduced GO after combination of chemical and thermal reduction reaches its lowest value of 4.20 eV, due to the presence of heteroatoms which doped the surface.