Indole-3-acetaldoxime (IAOx) is a branch point compound of tryptophan (Trp) metabolism in glucosinolate-producing species such as Arabidopsis, serving as a precursor to indole-glucosinolates (IGs), the defense compound camalexin, indole-3-acetonitrile (IAN) and indole-3-acetic acid (IAA). We synthesized [²H₅] and [¹³C₁ₒ¹⁵N₂]IAOx and [¹³C₆], [²H₅] and [2',2'-²H₂]IAN in order to quantify endogenous IAOx and IAN in Arabidopsis and tobacco, a non-IG producing species. We found that side chain-labeled [2',2'-²H₂]IAN overestimated the amount of IAN by 2-fold compared to when [²H₅]IAN was used as internal standard, presumably due to protium–deuterium exchange within the internal standard during extraction of plant tissue. We also determined that [¹³C₁]IAN underestimated the amount of IAN when the ratio of [¹³C₁]IAN standard to endogenous IAN was greater than five to one, whereas either [²H₅]IAN or [¹³C₆]IAN showed a linear relationship with endogenous IAN over a broader range of concentrations. Transgenic tobacco vector control lines did not have detectable levels of IAOx or IAN (limit of detection 100 pg/g fr. wt), while lines expressing either the IAOx-producing CYP79B2 or CYP79B3 genes from Arabidopsis under CaMV 35S promoter control accumulated IAOx in the range of 1–9 μg/g fr. wt. IAN levels in these lines ranged from 0.6 to 6.7 μg/g fr. wt, and IAA levels were 9–14-fold above levels in control lines. An Arabidopsis line expressing the same CYP79B2 overexpression construct accumulated IAOx in two of three lines measured (200 and 400 ng/g fr. wt) and accumulated IAN in all three lines. IAN is proposed to be a metabolite of IAOx or an enzymatic breakdown product of IGs induced upon tissue damage. Since tobacco does not produce detectable IGs, the tobacco data are consistent with IAN being a metabolite of IAOx. IAOx and IAN were also examined in the Arabidopsis activation tagged yucca mutant, and no accumulation of IAOx was found above the limits of detection but accumulation of IAN (3-fold above wt) occurred. The latter was surprising in light of recent reports that rule out IAOx and IAN as intermediates in YUCCA-mediated IAA synthesis.