Mitragyna speciosa or Kratom is a typical plant from West Kalimantan, Indonesia, and also is a plant endemic to Southeast Asia. Kratom contains thirty-seven alkaloid compounds. The major alkaloid compounds are mitragynine and 7-hydroxy mitragynine. Both have potent opioid effects due to their affinity for μ, K, and δ-opioid receptors. Mitragynine has a substantial opioid impact to create an analgesic effect, more potent than morphine and having a hypnotic effect at large dose. This plant has the potential to be abused. The United Nations Office on Drugs and Crime (UNODC) classifies kratom as a New Psychoactive Substances (NPS) in the plant-based group. An HPLC-Photodiode Array method was developed, optimized, and validated to quantify mitragynine and 7-hydroxymitragynine in kratom raw materials and finished products. Analysis mitragynine and 7-hydroxymitragynine performed using C18 Acquity® Bridged Ethylene Hybrid (BEH) column (1.7 μm, 100 mm x 2.1 mm); mobile phase combination were water, methanol, acetonitrile, and ammonium bicarbonate buffer 5mM in water pH 9.5; flow rate 0.3 mL/minute; a photodiode array detector at a wavelength of 225 nm and 219,9 nm for mitragynine and 7-hydroxymitragynine. The present study has fulfilled the UNODC validation parameter requirements. The method provides sensitive and selective analysis using HPLC-PDA with a calibration range for mitragynine was 0.1–5.0 μg/mL with correlation coefficients of 0.9997%. And for 7-hydroxy mitragynine was 0.25–1.0 μg/mL with correlation coefficients of 0.9999%. The method detection limits for mitragynine and 7-hydroxy mitragynine were 0.0021 and 0.0234 μg/mL, and also LoQ 0.0662 and 0.1665 μg/mL, respectively for mitragynine and 7-hydroxy mitragynine.