The aims of this study were to determine if histatin-1 (H1) is present in normal human tears and whether tear levels of H1 varied between normal patients and those with aqueous deficient dry eye disease (ADDE). Patient samples were obtained from 11 normal patients and 11 severe ADDE patients. Relevant patient characteristics, including age, sex, and dry eye disease (DED) diagnostic parameters were collected. Multiple qualitative and quantitative methods were used to compare the concentration of H1 between patient groups. Mixed linear modeling was used to compare H1 levels between groups, and diagnostic performance was assessed using the receiver-operator-characteristic (ROC). ADDE patients had significantly lower H1 concentrations (85.9 ± 63.7 ng/ml) than the normal group (891.6 ± 196.5 ng/ml) (p < 0.001), while controlling for age and sex. ROC analysis indicated that H1 concentration is potentially a biomarker for ADDE (area under curve = 0.96). Reclassification of patients by DED parameters including, Ocular Surface Disease Index (OSDI) (≤13, >13) and Schirmer I (without anesthesia) (<10 mm, ≥10 mm) showed significant differences in H1 level (OSDI, p = 0.004) and Schirmer I ((p = 0.010). In conclusion, this is the first preliminary report of the presence of H1 in human tears. H1 concentrations are lower in ADDE patients and H1 may have diagnostic potential in evaluation ADDE patients.