Articular cartilage covers the temporomandibular joint (TMJ) and provides smooth and nearly frictionless articulation while distributing mechanical loads to the subchondral bone. The thickness of the cartilage is considered to be an indicator of the stage of development, maturation, aging, loading history, and disease. The aim of our study was to develop a method for ex vivo assessment of the thickness of the cartilage that covers the TMJ and to compare that with two other existing methods. Eight porcine TMJ condyles were used to measure cartilage thickness. Three different methods were employed: needle penetration, micro-computed tomography (micro-CT), and histology; the latter was considered the gold standard. Histology and micro-CT scanning results showed no significant differences between thicknesses throughout the condyle. Needle penetration produced significantly higher values than histology, in the lateral and anterior regions. All three methods showed the anterior region to be thinner than the other regions. We concluded that overestimated thickness by the needle penetration is caused by the penetration of the needle through the first layer of subchondral bone, in which mineralization is less than in deeper layers. Micro-CT scanning method was found to be a valid method to quantify the thickness of the cartilage, and has the advantage of being non-destructive.