Tilapia (Oreochromis niloticus) scales represent an underutilized by-product with considerable potential as a source of bioactive peptides that can be released through enzymatic hydrolysis. This study evaluated the production of protein hydrolysates from demineralized tilapia scales using Alkaline Protease or Esperase 8.0 L^® (Alkaline Protease and Esperase 8.0 L® were kindly provided by Novozymes, Bagsværd, Denmark), and examined the impact of simulated gastrointestinal digestion (SGID) on their bioactivities. The highest degree of hydrolysis (DH) was obtained with Alkaline Protease (12.4%), generating peptide fractions predominantly below 1000 Da, with a major population around 888 Da. Both hydrolysates exhibited antioxidant activity, with the Alkaline Protease hydrolysate showing higher ferric reducing antioxidant power (FRAP) and Fe(II)-chelating activity. The hydrolysates also displayed significant angiotensin-converting enzyme (ACE) inhibitory activity (IC₅₀: 13–14 µg/mL), dipeptidyl peptidase IV (DPP-IV) inhibitory activity (IC₅₀: 0.66–0.69 mg/mL), and prolyl endopeptidase (PEP) inhibitory activity (IC₅₀: 0.63–0.72 mg/mL). The digests were non-cytotoxic at the concentrations tested (<10 mg/mL) in STC-1 enteroendocrine cells. Following SGID, increased ACE-inhibitory activity was observed, with IC₅₀ values as low as 4.6 µg/mL, whereas DPP-IV and PEP-inhibitory activities decreased. The intestinal digest of the Esperase hydrolysate also exhibited significant cellular antioxidant activity in the ROS assay. Overall, these results indicate that tilapia scale hydrolysates are a promising source of peptides associated with in vitro enzyme inhibitory and antioxidant activities. However, the specific bioactive peptides responsible for these effects were not identified. Therefore, further studies involving peptide characterization, bioavailability assessment, and in vivo validation are required to establish their physiological relevance and potential applications as functional ingredients.