Objective: Evaluating the presence of possible malignant disease in women with ovarian masses relies on medical imaging and serum marker findings. This study considers the role of serum Human Epididymal Protein 4 (HE4) antigen in combination with other serum markers to more effectively estimate the risk of malignancy in patients with isolated pelvic masses. Methods: We used prospectively collected biospecimens held by the Australian Ovarian Cancer Study (AOCS). Serum samples of patients with FIGO stage 1 epithelial ovarian cancer or with a benign condition were analysed for levels of circulating HE4 antigen, CA 125, and CEA, and test results were used to predict the presence of malignancy and to differentiate benign from malignant pelvic masses. Results: HE4 levels were significantly elevated amongst postmenopausal women and amongst patients with malignancy compared to premenopausal women and those with benign disease (p