Enzymatic isolation of chondrocytes from a cartilage biopsy is the first step to establish in vitro models of chondrogenesis or to generate cell-based grafts for cartilage repair. Such process is based on manually operated procedures and typically results in yields lower than 20% of the total available cells. In this study, we hypothesized that, as compared to conventionally used protocols, the enzymatic digestion of human articular cartilage in the presence of ascorbic acid 2-phosphate (AscA2P) or of sodium chloride (NaCl), in combination with the use of a perfusion bioreactor system, leads to a higher and more reproducible yield of cell populations with high proliferation and chondrogenic capacity. The addition of AscA2P within the enzymatic digestion medium did not significantly increase the cell yield, but resulted in a significant decrease of the intradonor variability in cell yield (-17.8% ± 10.7%, p = 0.0247) and in a significant increase of the proliferation rate of the isolated chondrocytes (+19.0% ± 1.4%, p < 0.05) with respect to the control group. The addition of NaCl during cartilage digestion did not modulate cell yield. When the cartilage digestion was further performed under direct perfusion flow, beneficial synergistic effects were achieved, with an overall increase of 34.7% ± 6.8% (p < 0.001) in the cell yield and an average decrease of 57.8% ± 11.2% (p < 0.01) in the coefficient of variation with respect to the control group. Importantly, by implementing this strategy it was possible to retrieve clonal subpopulations more efficiently capable of undergoing chondrogenesis, both in vitro and in vivo. Our findings bear relevance for the preparation of human chondrocytes for laboratory investigations, and in the perspective of efficient and streamlined manufacturing of cell/tissue grafts for articular cartilage repair.