We report the determination of the thickness of graphene layers by Auger electron spectroscopy (AES). We measure AES spectra of graphenes with different numbers of layers. The AES spectroscopy shows distinct spectrum shape, intensity, and energy characteristics with an increasing number of graphene layers. We also calculate electron inelastic mean free paths for graphene layers directly from these measurements. The method allows unambiguous and high-throughput determination of thickness up to six graphene layers and detection of defect and dopant in graphene films on almost any substrate. The availability of this reliable method will permit direct probing of graphene growth mechanisms and exploration of novel properties of graphenes with different thicknesses on diverse substrates.