Even if different LC-MS/MS methods for the determination of a number of steroids from serum, plasma, dry-blood spot and urine samples has been already described, in this work for the first time we present a robust and sensitive LC-MS/MS method that allows us to analyse seven tear steroids by performing only one extraction procedure from tears collected on Schirmer strip . We have demonstrated good sensitivity since we are able to detect up to 0.3 ng/mL for CORT, 0.05 ng/mL for 11-DECOL, ADIONE, 17-OHP and PROG, and 0.02 ng/mL for TESTO in tear samples. It can be an advantage since to date, the clinical used immunoassay for serum sex hormones have not been able to reliably measure the low concentrations, especially regarding TESTO in females and children . It is also to consider that usually in clinical practice, it is needed one assay for each steroid of interest, while LC-MS/MS methods give the opportunity to monitor multiple analytes in a single analysis. Therefore, to test the seven steroids in tears by using the conventional immunoassay methods it would be necessary to perform seven different sample collection. The methodology described involves a simple extraction of target steroids (CORT, CCONE, 11-DECOL, ADIONE, TESTO, 17-OHP and PROG) from tears collected on Schirmer strip to obtain a specific profile through the multiple reaction monitoring (MRM) acquisition mode for each analyte using two transition (quantifier and qualifier ions) to ensure lack of interferences. Thus, our LC-MS/MS method proved to have good specificity, sensitivity, and also good linearity and intra- and inter-assay imprecision. The use of LC-MS/MS allowed us to obtain chromatographic resolution of the analytes of interest, even for CCONE and 11-DECOL, which have the same nominal molecular mass. Unfortunately linearity for CCONE was not achieved.