Background: To prevent hemorrhage, fibrin clot formation, and subsequent activation of the inflammatory response, surgical preparation for articular cartilage regeneration should avoid penetration of the subchondral bone plate. Hypothesis: Current surgical procedures with ring curettes do not violate the subchondral bone plate. Study Design: Controlled laboratory study. Methods: The subchondral bone plates of normal human (n = 5) or sheep (n = 12) cadaver femoral condyles were prepared in vitro using either traditional debridement for autologous chondrocyte implantation/transplantation (ACI/ACT) or a modified approach aimed at deliberately violating the subchondral bone plate (ie, brute force). Effects were analyzed by light microscopy. In addition, subchondral bone plates of osteoarthritic (OA) human knees undergoing total knee arthroplasty (n = 5) or normal sheep knees (n = 5) were prepared in vivo. To approximate normal/regular wear, in humans, only OA samples with maximally grade 3A (International Cartilage Repair Society score) were used. Results: In both human and sheep (in vitro), no penetration of the subchondral bone plate was observed by either standard preparation or brute force. In vivo, standard preparation of human or sheep knees also did not violate the tidemark line. Human and sheep specimens prepared by brute force, however, occasionally showed areas with an open bone marrow space. Conclusion: Traditional debridement techniques for ACI/ACT using a ring curette do not violate the normal subchondral bone plate in vitro or in vivo. Even in OA knee joints, the bone plate is only violated by brute force. Therefore, the standard technique appears suitable for studies on cartilage regeneration in cases of traumatic and possibly even osteoarthritic defects. Clinical Relevance: The described surgical preparation technique is the traditional debridement technique for the ACI/ACT. The classic indication for the ACI/ACT is an articular cartilage injury, common in athletes.