Abstract Background Advancements in cartilage tissue engineering have the potential to ameliorate facial and joint reconstructive surgery as we know it. The translation of in vitro models of cartilage regeneration into clinical scenarios is the next phase of cartilage tissue engineering research. To engineer larger, more robust, and clinical relevant constructs, a great number of viable chondrocytic cells are needed. However, there is a paucity of literature concerning the most favorable method of chondrocyte isolation. Isolation methods are inconsistent, resulting in small yields and poor cell quality, and thus unreliable neocartilage formation. This study aimed to optimize the chondrocyte isolation protocol to give a maximum yield with optimal cell viability for the engineering of large cartilaginous constructs such as the human nose and ear. Methods We employed several enzymes (pronase, dispase, hyaluronidase, and collagenase), enzyme concentrations, and digest lengths to digest freshly harvested ovine nasoseptal cartilage. We used automated trypan blue live/dead staining, immunofluorescent labeling of CD44, collagenase II, collagenase I, and Aggrecan, and alamarBlue to assess cell yield and viability. Results Incubation length in enzymatic solutions had the greatest effect on cell viability, whereas concentrations of enzymes had a lesser effect. Isolated cells maintained their expression of chondrocyte-specific cell surface markers. Conclusions The optimum incubation period was 10 h using collagenase at a 0.2% (w/v) solution. An average of 1–1.5 × 106 cells could be harvested per gram of cartilage using this method.