Many chronic diseases result in organ failure, leaving organ transplantation as the only definitive therapeutic option. However, the severe shortage of donors has led to evaluate alternative approaches such as liver cell therapy (LCT). Lack of enough numbers of high-quality human hepatocytes has limited the outcome of LCT. In this sense, hepatocyte-like cells (HLCs) differentiated from induced pluripotent stem cells (iPSCs) are a promising alternative to primary hepatocytes since they can recapitulate hepatocyte properties and may avoid immune rejection, although their phenotype is still more fetal. Despite many different protocols for improving HLCs differentiation are under investigation, we have focused on exploring different matrices and assessing their contribution to the differentiation process. We have differentiated iPSCs into HLCs using four different culture surfaces Matrigel, Geltrex, LN111 and LN521, and have comparatively evaluated the level of differentiation. In addition to analyse the hepatic phenotype for its potential translation to the clinic, it is essential to ensure the loss of pluripotency markers. A decrease in pluripotency markers was observed in HLCs of all matrices as differentiation progressed, with minimal expression at the end of differentiation. Additionally, HLCs cultured in LN521 exhibited a higher expression of hepatic markers and increased urea and albumin production compared to the other matrices. Metabolic activity of phase I (CYP450) and phase II enzymes was also higher in HLCs differentiated on LN521.Therefore, our results have shown an improvement of the differentiation of iPSCs into HLCs when cells are differentiated on LN521 that could benefit they application in LCT.