Pefabloc and Trasylol are serine protease inhibitors that have been used during islet isolation to inhibit endogenous proteases (trypsin, chymotrypsin, and elastase) and improve islet yields. Using in vitro studies, we evaluated the effects of Pefabloc and Trasylol on the activities of these proteases and the effect of Pefabloc on islet function. In addition, it has been reported that Protector Solution (PS) enhances the efficiency of Pefabloc. Thus, we evaluated the efficacy of Pefabloc in the presence or absence of PS. EnzCheck protease assay was used to measure the activities of trypsin, chymotrypsin, elastase, liberase, and thermolysin in the presence or absence of 0.4 mmol/L Pefabloc (with or without PS) or 0.43 micromol/L Trasylol. We also tested switch samples containing the highest concentration of enzymes. Pefabloc significantly inhibited trypsin, chymotrypsin, elastase, and switch, but not liberase or thermolysin. Trasylol significantly inhibited all enzymes except for elastase and switch sample. Unexpectedly, the potency of Pefabloc was abrogated when diluted first in PS. Insulin release was diminished when islets were incubated or perifused with Pefabloc. In conclusion, Pefabloc when added appropriately significantly blocked in vitro protease activity. Unfortunately, Pefabloc also decreased islet insulin secretion, making it unsuitable for islet isolation. Trasylol cannot be used with collagenase because it impaired both liberase and thermolysin.