Background and Aims: Therapeutic approaches of iPS-derived hepatocytes are currently limited by undefined hepatic differentiation protocols. Because the iPS cell expansion and differentiation protocols is more often associated with animal-derived based extracellular matrix such as feeder layer cell, matrigel, serum supplement are largely undefined and raise a concerns of immunogenicity and microbial and viral contamination in clinical setting and high batch to batch variation in laboratory setting. Methods: To over these limitations, we integrate the nanostructured self-assembling peptides as a defined extracellular matrix for efficient hepatic differentiation of non-viral mini circle DNA based iPS cells in a clinically relevant bioreactor. We aim to establish and optimize a defined sequential differentiation protocol with growth factors, hormones, and cytokines in a time- dependent manner mimicking their secretion pattern during in vivo development of the liver to produce a clinically realistic application of iPS cells. In parallel, we evaluate potentiality of the sequential differentiation protocol with conventional cocktail exposure protocols. Results: We observed significantly more efficient hepatic functions upon sequential exposure rather than cocktail exposure. Additionally, we encapsulated the non-viral iPS derived hepatocytes using the same nanostructured self-assembling peptides and showed the albumin and urea performance per cell is almost similar to that in vivo. Conclusions: We established a chemically defined, efficient, scalable and inexpensive differentiation methodology for hepatic differentiation using nanostructured self-assembling peptides and generate clinical grade hepatocyte cells for effective therapeutic approaches. The author has none to declare.