Nothapodytes nimmoniana is a medicinally important plant producing camptothecin (CPT) which is a well-known anti-cancer drug. Its analogues viz. topotecan and irinotecan are currently used to treat variety of can-cers like lung, colon, ovarian cancers. The CPT is a monoterpene indole alkaloid class of compound and syn-thesised by the combination of terpene and indole pathway. Secologanin from terpene pathway and tryptamine from indole pathway condense together to form strictosidine which is a precursor of CPT. In this study, four different tissues viz. root, leaf, bark and fruit of N. nimmoniana were subjected to LC-QTOF-MS analysis. Total sixteen intermediate compounds in the CPT biosynthetic pathway with their relative accumu-lation were detected and confirmed by tandem mass spectrometry analysis. Two or more than two isomers of post-strictosidine pathway intermediates like deoxypumiloside, pumiloside and strictosamide, strictosa-mide epoxide were detected in roots and bark. Accumulation pattern of intermediate metabolites in roots suggests active biosynthesis of CPT in roots. Moreover, unlike Camptotheca acuminata (another CPT produc-ing plant), presence of both carboxylic acid derivatives and methyl esters of iridoid pathway intermediates such as loganic acid/ loganin, secologanic acid/ secologanin and strictosidinic acid/ strictosidine suggests existence of two or more interwoven pathways leading to CPT biosynthesis in N. nimmoniana.(c) 2023 SAAB. Published by Elsevier B.V. All rights reserved.