Since tears have already revealed important insights for studying eye disorders , and considering our experience in the development of liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the determination of metabolites related to pathologies , we aimed to measure steroid levels directly on tear samples, for the first time. Thus, a robust, specific, and selective method was developedfor the simultaneous quantification of the following steroids: cortisol (CORT); corticosterone (CCONE); 11-deoxycortisol (11-DECOL); 4-androstene-3,17-dione (ADIONE); testosterone (TESTO); 17α-hydroxyprogesterone (17-OHP); and progesterone (PROG). The proposal methodology was applied for the analysis of a small casuistry of tear samples from 14 female dry eye patients and 13 female controls.