In summary, this work revealed for the first time that tears represent a precious source of information for the study of the pathophysiologic state of the eye. In particular, in this pilot study steroid tear levels were measured and correlated to DED. Even if only a small cohort of tear samples were analysed, our innovative LC-MS/MS application added evidence of steroid level alteration in the tears of DED patients. Since the exact relationship between serum and tear steroid levels remains unknown, it could be interesting to study these two biological fluids from a large cohort of DED patients.