Background/aim: The purpose of this study is to determine the accuracy of bilateral inferior petrosal sinus sampling (IPSS) in lateralization and to investigate variables associated with accurate IPSS lateralization prediction. Materials and methods: Initially, data from 55 patients who underwent IPSS in our institution were reviewed retrospectively. IPSS lateralization and pituitary magnetic resonance imaging (MRI) results of these patients were compared with postoperative follow-up and immunohistochemical data to calculate the positive predictive values (PPVs) for IPSS and MRI. Variables likely to be associated with the accurate prediction of IPSS lateralization were analyzed. Results: Twenty-seven patients (85.2% female, mean age of 38.5 ± 13.1 years) were enrolled in the study. With IPSS, interpetrosal ratios were found to be ≥ 1.4 in 26 (96.2%) cases, and this ratio correctly predicted adenoma localization for 18 patients (PPV: 69.2%). For 16 (59.2%) patients, right lateralization was detected, while left lateralization was detected for 10 (37%) patients. Right-sided IPSS lateralization was associated with enhanced accuracy (p = 0.026). No masses were detected in the MRI images of 10 (37%) patients, while microadenoma of ≤ 6 mm was detected for 17 (63%) patients. MRI results (when positive) correctly identified adenoma localization for 14 of the patients with lateralization accuracy higher than that of IPSS (PPV: 82.3% vs. 69.2%). Conclusion: IPSS is a valuable procedure in detecting tumor lateralization, especially in patients with Cushing’s disease who have negative pituitary MRI results. However, since lateralization has a limited reliability, the pituitary gland should be comprehensively evaluated by taking into account the MRI findings (if positive) as well as data on the side of IPSS lateralization.