In order to define data distribution in each group, data matrix was statistically processed performing D’Agostino and Pearson omnibus normality test. Once normality was accepted, Student’s t-test was performed, otherwise the Mann Whitney test was carried out to assess the significantly expressed variables between the groups. Response levels were measured for all the seven steroids. CORT, ADIONE, and 17-OHP response levels resulted significantly decreased (p-value < 0.05) in dry eye patient samples compared to control samples. CCONE response level also resulted decreased in this comparison, although for this steroid we did not achieve the optimal conditions after the validation assay procedure. Figure 2 shows tear steroid profiling of dry eye and in healthy control patients (panel A). Data are mean values and bars represent the corresponding standard deviations (SD): ** indicates p < 0.01, and *** indicates p < 0.001. In Figure 2, panel B–D represent tear sample distribution for CORT, ADIONE and 17-OHP response level, respectively. In order to evaluate the diagnostic predictive power, we performed receiver operating characteristic (ROC) curve based on the three significantly different steroids obtained. After this investigation, the area under the curve (AUC) was 0.964, highlighting good sensitivity and specificity in discriminating the two study groups (Figure 3, panel A). We also performed the 100 cross-validation showing the predicted class probabilities of each sample, underlying good predictivity of the proposed model (p < 0.01) in discriminating dry eye patients (class 1) from healthy people (class 0) (Figure 3, panel B,C) .