This double-blind study examined effects of a protease enzyme blend (Prohydrolase®) added to whey protein on post-resistance exercise aminoacidemia and intramuscular anabolic signaling were investigated in ten resistance-trained males. Participants completed 4 sets of 8−10 repetitions in the leg press and leg extension exercises at 75% of 1-repetition maximum. Participants then consumed either 250 mg of Prohydrolase® + 26 g of whey protein (PW), 26 g whey alone (W), or non-nutritive control (CON) in counterbalanced order. Blood samples were obtained prior to exercise (baseline) and then immediately-post (IP), 30-, 60-, 90-, 120-, and 180-min post-exercise. Muscle biopsies were taken at baseline, 1-h (1H), and 3-h (3H) post-exercise. Phosphorylation of AKTSer437 was decreased (3H only: p < 0.001), mTORSer2448 was increased (1H: p = 0.025; 3H: p = 0.009), and p70S6KThr412 remained unchanged similarly for each condition. Plasma leucine, branch-chained amino acids, and essential amino acid concentrations for PW were significantly higher than CON (p < 0.05) at 30 min and similar to W. Compared to IP, PW was the only treatment with elevated plasma leucine levels at 30 min (p = 0.007; ∆ = 57.8 mmol/L, 95% Confidence Interval (CI): 20.0, 95.6) and EAA levels at 180 min (p = 0.003; ∆ = 179.1 mmol/L, 95% CI: 77.5, 280.7). Area under the curve amino acid analysis revealed no differences between PW and W. While no different than W, these data indicate that PW was the only group to produce elevated amino acid concentrations 30-min and 180-min post-ingestion.