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Objective(s): Avocado/soybean unsaponifible (ASU) possesses properties including chondroprotective, anticatabolic, and anabolic. The goal behind this research was to detect the effect of ASU and TGF-β3 on the chondrogenesis of human adipose-derived stem cells (hADSCs) on poly (lactic-co-glycolic) acid (PLGA)/hyaluronic acid (PLGA/HA) hybrid scaffold. Materials and Methods: First hADSCs were seeded in PLGA/Hyaluronic acid scaffold and cultured in chondrogenic media. These cells were assigned into 4 groups: control, TGFβ-3, ASU, and TGFβ-3+ASU. The viability was assessed separately by MTT. Real-time PCR was used to quantify the expression of chondrogenic specific genes [Sox9, collagen type II (ColII), Aggrecan (AGG)] and collagen type X (ColX). Moreover, Western blotting was employed to evaluate protein expression levels of collagens type II and X. Results: These findings indicated a significant increase in the proliferation and survival of hADSCs differentiated cells by ASU compared with the control group (P=0.008). Real-time PCR results revealed significant differences in the expression of AGG, SOX9, ColII, and ColX genes in the control group when compared with other groups (ASU, TGF-β3, and TGF-β3+ASU). ColII protein production significantly dropped in the TGF-β3 group in comparison with the TGF-β3+ASU group (0.000). The ColII (P=0.002) and ColX (P=0.002) protein production proved significantly higher in the TGF-β3+ASU group compared with the ASU group. Conclusion: Using the synergist form TGFβ-3, ASU induces chondrogenesis in hADSCs in PLGA/HA composite scaffold. This can be deduced with reduction of special markers of hyaline cartilage in comparison with ASU and decreased hypertrophic marker compared with TGF-β3. [ABSTRACT FROM AUTHOR]