Chelidonium majus is a well-known medicinal plant of papaveraceae family which demonstrates numerous 16 therapeutic effects due to the presence of isoquinolide alkaloids such as sanguinarine and berberine. In this study, after validation of an HPLC method, levels of these alkaloids in root samples of this plant collected from north of Iran were measured, and environmental factors affecting the amount of these compounds were also investigated. In this investigation, 9 groups of plants from different spots of north Iran were collected, and concentration levels of these alkaloids were measured applying a validated High Performance Liquid Chromatography- Photo diode Array/Mass Spectrometry (HPLC-PDA/MS) method. Then, the impact of geographical parameters encompassing altitude, average temperature, longitude and latitude coordinates on alkaloid contents was assessed. The validated method was selective, with good resolution, excellent linearity (r2> 0.99), high accuracy, sensitivity and precision. Also, the results illustrated that there was a direct correlation between longitude and altitude with the amount of alkaloids in plant, which means the more the levels of these variables, the more the amount of mentioned alkaloids. In an opposite manner, levels of the alkaloids reversely correlated with latitude coordinates and temperature, in a way that decreasing these variables resulted in raising the amount of the alkaloids. The validated method is a simple, fast, accurate, precise and robust. Also the environmental factors can impact on alkaloid contents and higher levels of alkaloids were associated with lower temperatures, high longitude, less latitude, and more altitude from sea level.