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• The correctness and accuracy of Stoney's formula was experimentally investigated. • Stoney's formula is not proper for finding larger non-equibiaxial thin film stress. • The use of a single curvature in Stoney's formula is too much over-simplified. • 19.57% stress biaxiality increase makes 654.5% up of maximum and minimum difference. In this paper, the radii of curvature and the stresses of transparent photoelastic circular disks without/with coating of the silicon dioxide (SiO 2) thin film with three different thicknesses were respectively measured by Fizeau interferometer and photoelasticity. In all past research work, thin film stresses (TFSs) of only two directions were measured and the difference between them was determined. However, the real maximum TFS can never be found. In this paper, TFSs of a number of directions of the substrate were measured so that both the magnitude and direction of principal stresses can be calculated. The calculated principal stress difference (PSD) by Stoney's formula and the equivalent TFS calculated by the enhanced exposure theory of photoelasticity (EEToP) are called PSD-STONEY and ETFS-EEToP, respectively. By comparing the results of the PSD-STONEY and PSD-ETFS-EEToP, the maximum and minimum differences are 28.35 MPa (215.4%) and 9.520 MPa (28.55%), respectively. Furthermore, with an increase about 19.57% of stress biaxiality of TFS, the increase of maximum and minimum difference between the results of Stoney's formula and photoelasticity is 654.5%. Based on the experimental findings of this paper, Stoney's formula is not suitable for calculating larger non-equibiaxial thin film stress. The assessment method of TFS developed in this paper for Stoney's formula with an independent measurement of substrate stress by photoelasticity is novel and can be used to verify other forms of Stoney's formula. Part of this work has been presented at the 2016 SEM 2016 Annual Conference & Exposition on Experimental and Applied Mechanics —Orlando, 6 Jun–9 Jun 2016 with the title "Wei-Chung Wang, et al., Investigation of Non-equibiaxial thin film stress by Using Stoney Formula". [ABSTRACT FROM AUTHOR]