Measures to counteract proteolysis during sample preparation are widely used; among them, protein extraction at a basic pH (Tris pH 11.0), sample boiling in sodium dodecyl sulfate (SDS), extraction in denaturing lysis solutions and the use of proteinase inhibitors combined with some of these approaches. Here, we tested their efficiency under stringent conditions using a high proteinase (trypsin and a mixture of pancreatic proteinases) contamination and as substrate, streptokinase, a protein highly sensitive to proteolytic degradation. Total degradation was observed in Tris pH 11.0. There was an efficient inhibition for the pancreatic proteinases after boiling in 1% SIDS, 1% dithiothreitol (DTT), while trypsin inhibition was dependent on the enzyme-to-substrate ratio. A panel of 21 lysis solutions with variable concentrations of urea, thiourea and detergents was essayed for the ability to counteract proteolysis. In all solutions containing 7-9 m urea, detergents and proteinase inhibitors but not containing thiourea, there was a strong proteolysis. However, in all samples containing 2 m thiourea, proteolysis was inhibited. Moreover, inhibition was dependent on the thiourea concentration. According to these results, we are prompted to consider that the well-known benefits of incorporating thiourea into the lysis solution are a result of two factors, its efficiency in solubilizing proteins and the inhibition of the proteolysis of sensitive substrates; both contributing to the detection of a higher number of species in two-dimensional electrophresis (2-DE) gels.