Abstract To determine the Angiotensin-I Converting Enzyme (ACE) inhibitory activity of marine cryptides, different methods were tested. ACE inhibition was measured using two synthetic substrates (N-[3-(2-Furyl) acryloyl]-Phe-Gly-Gly (FAPGG) and N-Hippuryl-His-Leu hydrate salt (HHL)) and a natural one: angiotensin-I. The IC50 value (defined as the concentration of inhibitory molecule needed to inhibit 50% of the ACE activity) of the reference synthetic inhibitor captopril was in the nano-molar range (1.79-15.1nM) when synthetic substrates were used whereas it exhibited IC50 of micro-molar range (16.71µM) with angiotensin-I. We chose losartan, an antagonist of angiotensin-II receptor as negative control for the ACE inhibition. Losartan was also able to inhibit ACE whatever the substrate tested, with IC50 of micro molar range (17.13-146µM). We defined this value as a limit above which molecules are not showing in vitro ACE inhibitory activity. Val-Trp (VW), Val-Tyr (VY), Lys-Tyr (KY), Lys-Trp (KW), Ile-Tyr (IY), Ala-Pro (AP), Val-Ile-Tyr (VIY), Leu-Lys-Pro (LKP), Gly-Pro-Leu (GPL), Ala-Lys-Lys (AKK) and Val-Ala-Pro (VAP) were tested as inhibitors of ACE with synthetic and natural substrates. IC50 displayed were substrate-dependent. With FAPGG as substrate, IW, VAP, KY, IY, AP, AKK and VIY show IC50 value over IC50 value of losartan and should not be considered as inhibitors of ACE. VY, VW, KW and LKP exhibited IC50 value lower than IC50 value of losartan for all substrates tested and were thus considered as good candidates for effectively decrease hypertension. It appears that the comparison of IC50 is not consistent when IC50 values are obtained with different substrates and different methods. In vitro ACE inhibitory activity assays should always include various ACE substrates and references like captopril and a negative control to obtain data reliable to discriminate ACE inhibitory peptides.