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Increasing demand for glutaminase (GLS) due to high rates of glutamine metabolism is considered one of the hallmarks of malignancy. In parallel, cancer antigen 125 (CA-125) is a commonly used ovarian tumor marker. This study aimed to compare the roles of GLS and CA-125 in distinguishing between benign and malignant ovarian tumors. The research was conducted as a comparative study, enrolling 156 patients with ovarian tumors. Preoperative serum CA-125 and GLS levels were analyzed to evaluate their effectiveness in distinguishing between benign and malignant ovarian tumors. The results revealed that the mean levels of CA-125 and GLS were significantly higher in malignant ovarian tumors compared with benign ones (389.54 ± 494.320 vs. 193.15 ± 529.932 (U/mL) and 17.37 ± 12.156 vs. 7.48 ± 4.095 (μg/mL), respectively). The CA-125 and GLS cutoff points of 108.2 U/mL and 18.32 μg/mL, respectively, were associated with malignant ovarian tumors. Multivariate analyses showed that GLS had higher predictive capabilities compared with CA-125 (odds ratio 9.4 vs. 2.1). The accuracy of using GLS combined with CA-125 was higher than using CA-125 alone (73.1% vs. 68.8%). In conclusion, higher levels of CA-125 and GLS are associated with malignant ovarian tumors. GLS outperforms CA-125 in distinguishing between benign and malignant ovarian tumors. The combination of GLS and CA-125 demonstrated improved accuracy for distinguishing benign and malignant ovarian tumors when compared with using CA-125 alone., (© 2023 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)