Considering that two-dimensional (2D) molybdenum trioxide has acquired more attention in the last few years, it is relevant to speed up thickness identification of this material. We provide two fast and non-destructive methods to evaluate the thickness of MoO3 flakes on SiO2/Si substrates. First, by means of quantitative analysis of the apparent color of the flakes in optical microscopy images, one can make a first approximation of the thickness with an uncertainty of ±3 nm. The second method is based on the fit of optical contrast spectra, acquired with micro-reflectance measurements, to a Fresnel law-based model that provides an accurate measurement of the flake thickness with ±2 nm of uncertainty.