Isolated sphenoid sinus disease (ISSD) is where there is a group of pathologies characterized by inflammation in one or both sphenoid sinuses. Although computer tomography (CT)-based 3D reconstruction remains the gold standard among noninvasive approaches to ISSD diagnostics, no standardized techniques for direct intraoperative measurements of the sphenoid sinus volume in ISSD patients have been documented. We suggest a novel technique for the intraoperative measurement of the sphenoid sinus volume. Our technique is based on filling the sinus with 0.01% methylene blue solution after an endoscopic endonasal sphenoidotomy. The proposed technique was applied to 40 ISSD patients during surgery. Obtained intraoperative measurements were compared to noninvasive measurements from 3D reconstructions based on preoperative CT scans. Our results demonstrated that the obtained measurements did not exhibit significant differences exceeding 0.4 cm, with CT-scan-based measurements in 39 out of 40 cases ( < 10, Wilcoxon sign-rank nonparametric test), thus confirming the accuracy of the proposed technique. Disagreements between direct intraoperative and CT-based measurements in a single case have been attributed to the presence of remaining pathological masses in the sinus, which was further confirmed during the secondary check of the operated sinus. Accordingly, we suggest that the agreement between the CT-based and intraoperative volume measurements can be used as an indicator of the successful elimination of all pathological masses from the sinus without having to perform an adequate exposure of the entire sphenoid sinus to reduce intraoperative bleeding. The proposed technique is accurate and does not require the involvement of specialized intraoperative CT scanners and avoids additional radiation exposure for the patient during an additional postoperation CT scan to confirm the success of the surgery.