Caseins are the principal milk proteins and serve as an important source of bioactive peptides with diverse beneficial effects for human health, such as antihypertensive, immunomodulating, anti-inflammatory, and antithrombotic. The objective of this study was to optimize, using the design of the experiment, the production of bioactive peptides from α-casein applying microwave-assisted enzyme digestion (MAED). The optimal MAED conditions (time, temperature, and enzyme/protein ratio) were established for pepsin (digestion time 4 min, temperature 41°C, and E:P ratio 1:40) and trypsin (digestion time 10 min, temperature 37°C and E:P ratio 1:200.) enzymes. Digestion yields and the intensity of different bioactivities, i.e., antimicrobial, antioxidant, and acetylcholinesterase and αglucosidase inhibitory activities, evaluated by the HPTLC-bioassay technique were used as response variables. Trypsin-formed α-casein peptides showed antimicrobial, antioxidant, and acetylcholinesterase inhibition activities, while pepsin-formed showed antimicrobial, antioxidant, and α-glucosidase inhibition activities. These results demonstrate that MAED is a fast and effective technique for bioactive peptide production from casein proteins.