The root of Plumbago indica L. is commercially available in herbal markets in both crude and powdered forms. P. indica root is a key ingredient in numerous polyherbal formulations. However, P. indica has two closely related species, P. zeylanica L. and P. auriculata Lam. Since only P. indica is traditionally used in Thai polyherbal products, adulteration with other species could potentially compromise the therapeutic efficacy and overall effectiveness of these formulations. To address this issue, a MassARRAY iPLEX assay was developed to accurately identify and differentiate P. indica from its closely related species. Five single nucleotide polymorphism (SNP) sites-positions 18, 112, 577, 623, and 652-within the internal transcribed spacer (ITS) region were selected as genetic markers for species identification. The assay demonstrated high accuracy in identifying P. indica and was capable of detecting the species at DNA concentrations as low as 0.01 ng/µL. Additionally, the assay successfully identified P. zeylanica in commercial crude drug samples, highlighting potential instances of adulteration. Furthermore, it was able to distinguish P. indica in mixed samples containing P. indica, along with either P. zeylanica or P. auriculata. The developed MassARRAY iPLEX assay proves to be a reliable and effective molecular tool for authenticating P. indica raw materials. Its application holds significant potential for ensuring the integrity of herbal products by preventing misidentification and adulteration.