Adipose-derived stromal cells (ASCs) are usually isolated by digestion with collagenase. We have compared alternative methods to isolate ASCs in a more economically viable protocol. Nine protocols using red blood cells lysis buffer solution, trypsin, collagenase and centrifugation were compared; the isolation rate, cell viability, expansion rate, immunophenotype and differentiation in adipogenic and osteogenic lineages were analyzed. ASCs were isolated and successfully maintained by digestion with trypsin. Cells presented similar immunophenotypes, adipogenic differentiation and in vitro proliferation but an osteogenic differentiation capacity up to seven times higher than ASCs isolated by collagenase. This alternative protocol is thus efficient and more cost-effective than the commonly-used methods and may represent a promising protocol for obtaining ASCs for bone tissue engineering.