ONE OF THE BIOENGINEERING GOALS IS TO PRODUCE BIOMATERIALS THAT CAN REPLACE DAMAGED TISSUES. MANY DERMAL SUBSTITUTES ARE CURRENTLY AVAILABLE, BUT THE HIGH COSTS MAKE THEM LESS AFFORDABLE FOR THE GENERAL POPULATION. DECELLULARIZED SKIN PRESENTS A GREAT POTENTIAL AS A SKIN SUBSTITUTE. THE AIM OF THIS STUDY WAS TO DEVELOP A PROTOCOL FOR DECELLULARIZATION OF MURINE SKIN AND ANALYZE ITS STRUCTURE TO PRODUCE A DERMAL SUBSTITUTE. DISCARDED RAT SKIN (CEUA 32510) WAS DECELLULARIZED BY INCUBATION IN HYPERTONIC SOLUTIONS, TRITON X-100 AND TRYPSIN UNDER CONTINUOUS AGITATION AND THREE DIFFERENT INCUBATION TIMES WERE TESTED. THE GENOMIC DNA WAS QUANTIFIED AND COMPARED WITH NON-DECELLULARIZED SKIN. IN ORDER TO VERIFY THE EFFICIENCY, HISTOLOGICAL ANALYSES WERE PERFORMED. THE SAMPLES WERE SECTIONED ON MICROTOME IN 5μM THICKNESS AND STAINED WITH DAPI, MASSON’S TRICHROME AND HAEMATOXYLIN AND EOSIN (HE). THE DNA QUANTIFICATION ANALYSIS SHOWED THAT THE DECELLULARIZED SAMPLES PRESENTED A MUCH LOWER DNA CONTENT COMPARED TO CONTROL SKIN (3.026±1.06 AND 111.8±7.02 MG GDNA/MG TISSUE, RESPECTIVELY). THE HISTOLOGICAL SECTIONS STAINED WITH DAPI PRESENTED NORMAL NUCLEAR DISTRIBUTION OF THE CONTROL SKIN, BUT CELL NUCLEI WERE NOT DETECTED ON THE DECELLULARIZED SAMPLES. THE HE STAINING OF THE DECELLULARIZED SAMPLES EXHIBITED A CONSERVED MATRIX STRUCTURE, WITH THE MAINTENANCE OF THE DERMIS EXTRACELLULAR MATRIX. THE SAMPLES STAINED WITH MASSON’S TRICHROME SHOWED A STRUCTURE CONSISTING PREDOMINANTLY OF COLLAGEN. THUS, IT WAS POSSIBLE TO ESTABLISH AN EFFICIENT DECELLULARIZATION PROTOCOL OF THE RAT SKIN THAT CONSERVED THE SKIN EXTRACELLULAR MATRIX AND COLLAGEN CONTENT, BEING A PROMISING TECHNIQUE FOR THE PRODUCTION OF DERMAL SUBSTITUTES IN REGENERATIVE MEDICINE.