The objective of this study was to generate protein hydrolysates with dual in vitro inhibitory activities against dipeptidyl-peptidase IV (DPP-IV) and angiotensin-I converting enzyme (ACE), by proteolytic enzymes acting either individually or sequentially on steelhead skin gelatin. The results showed strong dual bioactivity in the low molecular weight (<3kDa) fractions obtained by ultrafiltration of hydrolysates produced using the enzymes pepsin, CorolaseN or papain. Alternatively, unfractionated hydrolysates exhibiting high ACE and DPP-IV inhibitory activities as well as yields could be attained by strategic selection of two enzymes for successive hydrolysis of gelatin. In particular, hydrolysates produced using 4% papain for 2h followed either by ultrafiltration or by a second hydrolysis with 1% CorolaseN for 2h, presented potent dual activity as ACE and DPP-IV inhibitors, and should be investigated further as potential functional food ingredients or nutraceuticals for the management of hypertension and diabetes.