2-Methoxypancracine is a montanine-type alkaloid exhibiting anti-inflammatory, antioxidant, and antibacterial effects. The bulbs of Hippeastrum reticulatum (L'Her.) Herb. contain large amounts of 2-methoxypancracine. The aim of this study was to develop a HPLC-DAD method to quantify 2-methoxypancracine content in H. reticulatum. The HPLC analysis was carried out using a Poroshell 120 EC-C18 column eluted with acetonitrile and 0.01% methylamine (35:65, v/v) mixture in water at a flow rate of 0.5 mL/min with detection at 205 nm. Method validation was performed to assure its linearity, precision, accuracy, as well as the limit of detection (LOD) and limit of quantitation (LOQ). 2-Methoxypancracine displayed a linear relationship within a range of 2.5 - 103 mu g/mL. The developed method was shown to be precise with relative standard deviation less than 2.5%. The average recovery of 2-methoxypancracine was 101.2. The LOD and LOQ values were calculated as 0.08 mu g/mL and 0.25 mu g/mL, respectively. The concentrations of 2-methoxypancracine in the alkaloid fraction and in the dried powder of H. reticulatum were 28.95%, and 0.0740%, respectively. The validated method provides a useful tool for the quantification of 2-methoxypancracine in H. reticulatum. This is the first report of significant 2-methoxypancracine content in H. reticulatum.