The use of animal products in the derivation and maintenance of human pluripotent stem cells (hPSCs) limits their possible applications in research and in clinics. Thus, one of the major goals in regenerative medicine is the establishment of animal-free conditions to support the culture and differentiation of human stem cells. Human fibroblasts produce an extracellular matrix (ECM) which can be extracted without the use of detergents, sterilized, and then used to coat tissue culture plates.We have shown that human embryonic stem cells (hESCs) grown on this matrix maintain their pluripotency in the presence of medium conditioned by fibroblast cells, and that these cells maintain expression of surface proteins (SSEA4, Tra1-60, Tra1-81), alkaline phosphatase activity, and specific intracellular markers (Nanog, Oct-4, Tert, FoxD3) in hESCs. This growth system reduces exposure of hPSCs to feeder layers and animal ingredients, thereby limiting the risk of pathogenic contamination and additionally, facilitating their manipulation. Herein we present an improved version of our previous protocol for extracting ECM from human foreskin fibroblast using a different buffer. Our new hypotonic shock method is detergent-free, reduces costs, and preserves the integrity of the extracted ECM. This improved protocol has been validated for undifferentiated-state hPSC maintenance (more than 40 passages), stem cell differentiation, and for cell migration assays.