Nicotinamide adenine dinucleotide (NAD⁺) is a core coenzyme in cellular energy metabolism and redox homeostasis, with pharmacological effects in cardiovascular and neurodegenerative diseases and in aging. Its non-clinical safety was evaluated through GLP-compliant studies that included safety pharmacology (single intramuscular injection, 3–30 mg/kg, Beagle dogs, telemetry), acute toxicity (single intramuscular injection, 45–180 mg/kg, SD rats), repeated-dose toxicity (10–100 mg/kg intramuscular, once daily for 4 weeks plus a 4-week recovery, SD rats), muscular irritation (intramuscular, 14 consecutive days, New Zealand rabbits), in vitro hemolysis (2% rabbit erythrocytes), and active systemic anaphylaxis (intramuscular sensitization, intravenous challenge, Hartley guinea pigs). In safety pharmacology, 3 mg/kg was the safe dose; 10 and 30 mg/kg caused increased heart rate, shortened PR and QT intervals, and decreased blood pressure (no significant difference in QTcF). The acute toxicity MTD was ≥ 180 mg/kg. The repeated-dose NOAEL was 100 mg/kg; local injection-site and sciatic-nerve lesions were reversible. Muscular irritation, in vitro hemolysis, and active systemic anaphylaxis tests were all negative. Overall, NAD⁺ showed a favorable non-clinical safety profile over the dose range tested, and the bioanalytical methods established here provide a basis for its further development and clinical application.