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To estimate the diagnostic performance and reference values of serum cancer antigen (Ca)15-3 levels in the triage of adnexal masses.This retrospective cohort study was carried out in 481 patients referred to the Gynecology Department at Carmel Medical Center due to adnexal mass between years 2005 and 2012. All patients underwent surgery with histopathologically confirmed diagnosis and routine preoperative measurements of serum Ca125 and Ca15-3.Combination of Ca125 with Ca15-3 elevated the sensitivity of Ca125 alone (from 86.9% to 93.2%; P=0.029), along with reduction of its specificity (from 80.5% to 69.5%; P=0.005) in differentiation between malignant and benign cases. According to receiver operating characteristic curve, Ca15-3 level of 21 U/mL was shown to be the optimal reference value for malignancy detection. All cases with Ca15-3 levels above 44.5 U/mL were malignant, mostly of primary ovarian source.As Ca15-3 assessment allowed detection of significantly more malignancy cases, we believe that measurement of this marker in combination with Ca125 is worthwhile in patients presenting with adnexal masses. The cutoff of 21 U/mL seems to be the optimal value in this specific population. High Ca15-3 levels (above 44.5 U/mL) strongly direct to a diagnosis of malignancy, mostly of primary ovarian tumors rather than breast malignancy.