Diseases that affect the cornea can lead to severe vision loss and have tremendous social impact. These diseases are associated to deviations from normal structural order and orientation of collagen fibril bundles. Unfortunately, resolving non-invasively the corneal collagen structure is not possible to date. In this work, polarization sensitive second harmonic generation (pSHG) microscopy is used to obtain information with molecular specificity on microstructure of human corneas. This information is used to develop a set of label-free imaging biomarkers that were generated by means of a novel methodology based on mathematical tensorial calculus. The method is proven to be highly sensitive and robust. The use of these biomarkers permits accurate characterization of the anisotropic, depth-dependent, structural organization of corneal collagen fibril bundles without any a priori information. The method can be valuable to improve understanding of microstructural pathophysiological changes of the human cornea close to in vivo conditions.