The ability to provide absolute calibrated measurement of the laryngeal structures during phonation is of paramount importance to voice science and clinical practice. Calibrated three-dimensional measurement could provide essential information for modeling purposes, for studying the developmental aspects of vocal fold vibration, for refining functional voice assessment and treatment outcomes evaluation, and for more accurate staging and grading of laryngeal disease. Recently, a laser-calibrated transnasal fiberoptic endoscope compatible with high-speed videoendoscopy (HSV) and capable of providing three-dimensional measurements was developed. The optical principle employed is to project a grid of 7 × 7 green laser points across the field of view (FOV) at an angle relative to the imaging axis, such that (after calibration) the position of each laser point within the FOV encodes the vertical distance from the tip of the endoscope to the laryngeal tissues. The purpose of this study was to develop a precise method for vertical calibration of the endoscope. Investigating the position of the laser points showed that, besides the vertical distance, they also depend on the parameters of the lens coupler, including the FOV position within the image frame and the rotation angle of the endoscope. The presented automatic calibration method was developed to compensate for the effect of these parameters. Statistical image processing and pattern recognition were used to detect the FOV, the center of FOV, and the fiducial marker. This step normalizes the HSV frames to a standard coordinate system and removes the dependence of the laser-point positions on the parameters of the lens coupler. Then, using a statistical learning technique, a calibration protocol was developed to model the trajectories of all laser points as the working distance was varied. Finally, a set of experiments was conducted to measure the accuracy and reliability of every step of the procedure. The system was able to measure absolute vertical distance with mean percent error in the range of 1.7% to 4.7%, depending on the working distance.