Craniofacial cephalometric analysis is a diagnostic tool used for the assessment of the relationship of various bones and soft tissues in the head and face. Cephalometric analysis has been traditionally conducted with the use of 2D radiographs and landmark sets and restricted to size, linear and angular measurements, and 2D relationships. The increasing use of 3D cone beam computed tomography (CBCT) scans in the dental field dictates the need for the evolution to 3D cephalometric analysis, which incorporates shape and a more realistic analysis of longitudinal development in all three planes. This study is a demonstration of 3D cephalometric analysis with the use of a validated set of skeletal tissue landmarks on human CBCT scans. Detailed instructions for the annotation of each landmark on a 3D volume are provided as part of a step-by-step protocol. The generated measurements and 3D coordinates of the landmarks can be exported and used both for clinical and research purposes. The introduction of 3D cephalometric analysis in basic and clinical craniofacial studies will lead to future advancements in the field of craniofacial growth and development.