The alkaloids from Cinchona ledgeriana shoot cultures and from Cinchona robasta shoot cultures and a compact globular structure (CGS) culture were analyzed by thermospray liquid chromatography/mass spectrometry (TSP LC/MS). Because of the relative stability of the alkaloids under TSP discharge ionization conditions, a protonated molecule was observed in the mass spectra with hardly any fragmentation. When the reference compounds were available, the knowledge of the molecular mass and of the retention time was sufficient to identify most of the alkaloids. HPLC with UV photodiode-array detection complemented LC/MS perfectly by providing information about the aromatic part of the alkaloids (structure and substitution pattern). New alkaloids detected in Cinchona in vitro cultures were 5 methoxytryptamine and corynantheal. In order to determine whether 5-methoxytryptamine was a precursor of the methoxylated quinolines, this indole was incubated with secologanin and several Cinchona robusta crude protein extracts. Under all conditions tested, the coupling of 5-methoxytryptamine with secologanin remained unsuccessful. Only tryptamine condensed with secologanm to yield strictosidine. These results indicate that Ginchona cells are able to methoxylate simple indoles like tryptamine and that 5-methoxytryptamine is very likely not used for the subsequent biosynthesis of the methoxylated quinolines.