Intraoperative optical coherence tomography (OCT) systems provide high-resolution, real-time visualization and/or guidance of microsurgical procedures. While the use of intraoperative OCT in ophthalmology has significantly improved qualitative visualization of surgical procedures inside the eye, new surgical techniques to deliver therapeutics have highlighted the lack of quantitative information available with current-generation intraoperative systems. Indirect viewing systems used for retinal surgeries introduce distortions into the resulting OCT images, making it particularly challenging to make calibrated quantitative measurements. Using an intraoperative OCT system based in part on the Leica Enfocus surgical microscope interface, we have devised novel measurement procedures, which allowed us to build optical and mathematical models to perform validation of quantitative measurements of intraocular structures for intraoperative OCT. These procedures optimize a complete optical model of the sample arm including the OCT scanner, viewing attachments, and the patient's eye, thus obtaining the voxel pitch throughout an OCT volume and performing quantitative measurements of the dimensions of imaged objects within the operative field. We performed initial validation by measuring objects of known size in a controlled eye phantom as well as ex vivo porcine eyes. The technique was then extended to measure other objects and structures in ex vivo porcine eyes and in vivo human eyes.