Background: Anatomical validation studies of cervical ultrasound images are sparse. Validation is crucial to ensure accurate interpretation of cervical ultrasound images and to develop standardized reliable ultrasound procedures to identify cervical anatomical structures. The aim of this study was to acquire validated ultrasound images of cervical bony structures and to develop a reliable method to detect and count the cervical segmental levels. Methods: An anatomical model of a cervical spine, embedded in gelatin, was inserted in a specially developed measurement device. This provided ultrasound images of cervical bony structures. Anatomical validation was achieved by laser light beams projecting the center of the ultrasound image on the cervical bony structures through a transparent gelatin. Results: Anatomically validated ultrasound images of different cervical bony structures were taken from dorsal, ventral, and lateral perspectives. Potentially relevant anatomical landmarks were defined and validated. Test/retest analysis for positioning showed a reproducibility with an intraclass correlation coefficient for single measures of 0.99. Besides providing validated ultrasound images of bony structures, this model helped to develop a method to detect and count the cervical segmental levels in vivo at long-axis position, in a dorsolateral (paramedian) view at the level of the laminae, starting from the base of the skull and sliding the ultrasound probe caudally. Conclusions: Ultrasound bony images of the cervical vertebrae were validated with an in vitro model. Anatomical bony landmarks are the mastoid process, the transverse process of C1, the tubercles of C6 and C7, and the cervical laminae. Especially, the cervical dorsal laminae serve best as anatomical bony landmarks to reliably detect the cervical segmental levels in vivo.