Aconitum heterophyllum Wall., a commercially important Himalayan medicinal plant, is used in various herbal formulations and has high market demand. Given its esteemed medicinal properties, considerable demand, and prioritized status, there’s a pressing requirement for thorough quality assessment methods to ensure high throughput. Genus Aconitum is also well known for its toxicity owing to the presence of diester diterpenoid alkaloids. Hence it is essential to develop a UHPLC-PDA-ELSD-MS based quantification method enabling simultaneous quantification of primary marker compounds alongside toxic diterpenoid alkaloids. The method showcased strong linearity, with detection and quantification limits covering a broad range. Precision was evident with consistently low relative standard deviations both within and across days. Furthermore, accuracy was demonstrated within acceptable bounds. Quantification results revealed the maximum content of atisinium and hetisine in hydroethanolic root extract [atisinium (≅78.88 mg/g) and hetisine (≅50.17 mg/g)] In addition, a network pharmacology-based approach has been applied to predict potential biological targets. The network pharmacology approach suggests the plant’s potential in treating neuralgia, renal fibrosis, diabetes mellitus, lung diseases, cancer, and more, emphasizing the need for scientific validation and indicating future research directions. Overall, the developed method represents a reliable and efficient quality assessment method for the simultaneous quantification of marker compounds and toxic diterpenoid alkaloids in the roots of A. heterophyllum. Network pharmacology predicts pharmacological potentials that require scientific validation highlighting future scope.