There has been emerging interest in exploring single-sheet 2D layered structures other than graphene to explore potentially interesting properties and phenomena. The preparation, isolation and rapid unambiguous characterization of large size ultrathin layers of MoS2, GaS, and GaSe deposited onto SiO2/Si substrates is reported. Optical color contrast is identified using reflection optical microscopy for layers with various thicknesses. The optical contrast of these thin layers is correlated with atomic force microscopy (AFM) and Raman spectroscopy to determine the exact thickness and to calculate number of the atomic layers present in the thin flakes and sheets. Collectively, optical microscopy, AFM, and Raman spectroscopy combined with Raman imaging data are analyzed to determine the thickness (and thus, the number of unit layers) of the MoS2, GaS, and GaSe ultrathin flakes in a fast, non-destructive, and unambiguous manner. These findings may enable experimental access to and unambiguous determination of layered chalcogenides for scientific exploration and potential technological applications.