The zygapophyseal (facet) joint plays a critical role in load transmission and stability of the spine, and facet degeneration is a common consequence of aging and osteoarthritis. The ability to accurately measure facet space is important, as decreased facet space is associated with facet degeneration and lower back pain. Although grading systems exist for assessing facet joint space narrowing, static imaging fails to characterize changes in the facet gap under load that play a role in segmental stability. Current methods for estimating the dynamic behavior of the facet joint are either inaccurate, radiation costly, or clinically impractical. In the current study, we demonstrate the feasibility of a novel method for 3D measurement of facet joint space using digital tomosynthesis (DTS) imaging in supine and standing positions. Facet gap measurements were found to be strongly correlated with (r to 0.98) and accurate (<​20 µm error for median facet gap) relative to microcomputed tomography reference values. In a pilot in vivo demonstration with seven participants, the effect of physiological loading was detectable, with median facet joint space being larger in standing as compared to supine images (p <​ 0.0001). The presented approach may be useful in directly characterizing changes in the facet joint relevant to segmental stability that are not readily assessed via current clinical imaging methods.