In conclusion, we present for the first time a robust, specific, and selective LC-MS/MS method for the simultaneous quantification of seven steroids from tear samples. The method was, also, applied to a small casuistry of tear samples from 14 female DED patients and 13 female controls, showing a significant reduction in tear response levels for CORT, ADIONE, and 17-OHP in DED patients respect to healthy people. After statistical investigation, our proposal model demonstrated a good predictive power in identifying DED patients. Our data confirm the idea that tears can be used as a precious source of information for the study pathophysiologic state of the eye and of other different disorders.