Purpose of investigation: HE4 (human epididymis protein 4) is suggested to be used as a potential new biomarker to identify ovarian malignancies from benign adnexal masses. The aim of this study was to evaluate HE4, in comparison with CA125 and Risk of Ovarian Malignancy Algorithm (ROMA) index in benign gynecological diseases and ovarian cancer, and additionally to determine the reference range for HE4 in healthy Turkish women. Materials and Methods: CA125 and HE4 serum levels were determined in 96 patients with benign gynecological diseases, 47 patients with ovarian cancer and 106 healthy women using a specific analyzer. CA125 and HE4 cut-offs were 35 U/ml and 70 pmol/L, respectively. Results: HE4 had significantly higher concentrations in ovarian cancer than benign gynecologic disorders (p < 0.005). Tumor marker sensitivity in ovarian cancer was 78% for HE4, 63% for CA125, and 88% for ROMA index at 95% specificity. A significantly higher area under the Receiver operator characteristic (ROC) curve was obtained with HE4 and ROMA index than CA125 in the differential diagnosis of benign gynecological diseases versus ovarian cancer (0.929, 0.955, and 0.781, respectively). Reference limits for serum HE4 in healthy Turkish women was determined as 28.9 - 62.4 pmol/L for premenopausal and 23.7- 152.4 pmol/L for postmenopausal women. Conclusions: In the diagnosis of ovarian cancer, HE4 had higher sensitivity, as a single tumor marker. The sensitivity of HE4 and ROMA index in postmenopausal women was higher than premenopausal women for detecting ovarian cancer.