Anthropometry is a method for quantifying body size and shape often used to derive body composition and health risk prediction models. Recent technology advancements led to development of three-dimensional (3D) optical scanners that can overcome most of the limitations associated with manual anthropometric data collection. However, each of the currently available devices offers proprietary measurements that do not match conventional anthropometric definitions. The aim of the current study was to develop and then evaluate the precision and accuracy of new "universal" 3D optical analysis software that calculates digital anthropometric volumes using identical standard landmarks across scanners. Dual-energy x-ray absorptiometry (DXA) and air displacement plethysmography (ADP) total body and regional volume and fat mass reference measurements and 3D optical scans from two proprietary devices were collected from 356 participants to evaluate the robustness of total body and regional volume and fat mass measurements calculated by the developed software. Linear regression modeling with threefold cross validation was used to evaluate total body and regional fat masses from 3D scans. Total body and regional volumes measured by DXA and ADP had strong associations with corresponding estimates from the commercial 3D optical scanners coupled with the universal software (e.g., R2 =0.98 for Styku and R2 =1.00 for SS20, for both DXA and ADP comparisons). Regional body volumes also had strong correlation between DXA and the 3DO scanners (e.g., for arm, leg and trunk, respective R2 s of 0.75, 0.86, and 0.97 for Styku and 0.79, 0.89, and 0.98 for SS20). Similarly, there were strong associations between DXA- measured total body and regional fat mass and 3D optical estimates calculated by the universal software (e.g., for total body, arm, leg and trunk, respective R2 s of 0.86, 0.72, 0.77, and 0.88 for Styku and 0.84, 0.76, 0.78, and 0.85 for SS20). Absolute differences in volumes and fat mass between the reference methods and the universal software values revealed underlying proprietary scanner differences that can be improved when designing future devices. The current study suggests that, when compared against values calculated using DXA and ADP, the universal software was able to measure total and regional body volumes reliably from scans obtained by two different scanners. The universal software, with future refinements, combined with potential optical scanner design improvements, creates new opportunities for developing large multicenter anthropometric databases with uniformly defined body dimensions that can be used for modeling health risks. Shape Up! Adults Study, NCT0363785.