This study reports tympanic cavity (TC) volume in newborns, which was missing in the literature. Ex vivo histology and computerized tomography (CT) scans were performed on temporal bone and data were analyzed in part using software developed in house. CT images with a slice thickness of 0.5 mm were obtained from 5 newborn cadavers and analyzed independently by two expert researchers. The border of the TC was delineated manually and measurement of area of interest was calculated on masked images. Then, the area measurements from all sections were added to estimate the total volume. The agreements between the histological and CT findings were then compared for accuracy, repeatability and reliability. The Dice and Jaccard similarity coefficient measures were used as a statistical validation metric to evaluate the assessor's performance in manual volume segmentation. Good assessor agreement was observed with average Dice values above 0.8 indicating that consistent and reliable volume measurements were feasible. The proposed protocol was shown to be accurate in calculating the TC volume, and thus can be used for computer-assisted presurgical planning or for diagnosing structural alterations in TC.