AbstractMolybdenum trioxide (MoO₃) in-plane anisotropy has increasingly attracted the attention of the scientific community in the last few years. Many of the observed in-plane anisotropic properties stem from the anisotropic refractive index and elastic constants of the material but a comprehensive analysis of these fundamental properties is still lacking. Here we employ Raman and micro-reflectance measurements, using polarized light, to determine the angular dependence of the refractive index of thin MoO₃ flakes and we study the directional dependence of the MoO₃ Young’s modulus using the buckling metrology method. We found that MoO₃ displays one of the largest in-plane anisotropic mechanical properties reported for 2D materials so far.