Nordihydroguaiaretic acid (NDGA), is a naturally-occurring lignan isolated from the creosote bush (Larrea tridentata). The aqueous extract of this shrub, commonly referred to as Chaparral tea, was listed in the American pharmacopeia as an ethnobotanical used to treat tuberculosis, arthritis and cancer. Other documented traditional applications of creosote bush extract include treatment for infertility, rheumatism, arthritis, diabetes, gallbladder and kidney stones, pain and inflammation among many others. In spite of the numerous pharmacological properties, NDGA use has been associated with toxicities including hepatotoxicity in humans. Previous studies in our group showed that oxidative cyclization of NDGA (a di-catechol) at physiological pH forms a dibenzocyclooctadiene that may have therapeutic benefits whilst oxidation to ortho-quinone likely mediates toxicological properties. In order to investigate the structural features responsible for pharmacological and toxicological properties, a series of NDGA analogues were designed, synthesized and characterized for the purpose of studying their oxidative metabolism. Literature procedures were modified to successfully prepare seven lignan analogues via multi-step synthesis. In our effort to understand the mechanisms of NDGA intramolecular cyclization, the prepared analogues were incubated under previously established conditions where NDGA autoxidized to yield the dibenzocyclooctadiene derivative. We also evaluated the stability of the analogues under the conditions of this study. Furthermore, we evaluated bioactivation potential of the prepared analogues with a goal of eliminating reactive metabolite liability through rational structural modification. We incubated NDGA and its analogues in rat liver microsomes (RLM) in the presence of glutathione as a nucleophilic trapping agent. Standards for comparison were generated by performing glutathione trapping experiments with chemical and enzyme oxidation systems. The potential of the dibenzocyclooctadiene lignan 2 derived from NDGA under physiological conditions to contribute to toxicological properties via reactive metabolite formation was also evaluated. Glutathione conjugates were detected by electrospray ionization-mass spectrometry (ESI-MS) scanning for neutral loss (NL) 129 Da or 307 Da in positive ion mode or precursor ion (PI) scanning for 272 Da in negative ion mode and further characterized by liquid chromatography–tandem mass spectrometry (LC–MS/MS) or in a single LC-MS run using multiple reactions monitoring (MRM) as a survey scan to trigger acquisition of enhanced product ion (EPI) data. We determined that NDGA autoxidation at pH 7.4 is dependent on substituents and/or substitution pattern on the two aromatic rings. In particular, spontaneous intramolecular cyclization to a dibenzocyclooctadiene required a di-catechol lignan, raising the possibility that o-Q formation may not be necessary for cyclization to occur. Cyclization was significantly inhibited in the presence of excess GSH which supports the involvement of free radicals as opposed to o-Q in the intramolecular cyclization process. The mono-catechol analogues A1 and A4 underwent oxidation to o-Q but no evidence of cyclization was found implying that electrophilic substitution cannot account for NDGA cyclization. The phenol-type analogues were oxidatively more stable in comparison with the catechol-type analogues at pH 7.4. The results demonstrate that electrophilic substitution makes no contribution to the intramolecular cyclization process and that a radical mediated process accurately describes the situation for NDGA. Oxidative metabolism and bioactivation studies on NDGA and its analogues revealed that reactive metabolites formation is dependent on substitution and/or substitution pattern of the aromatic rings. Cytochrome P450-mediated oxidation of NDGA and its catechol-type analogues yielded electrophilic intermediates which reacted with GSH. The GSH mono-conjugates were identified as ring adducts derived from o-Q although the position at which the GSH binds to the aromatic rings could not be determined. We also found that NL 129 or 307 scanning in positive ionization mode has potential diagnostic utility in distinguishing between aromatic and benzylic GSH conjugates although further studies may be required for validation. We found no evidence of p-QM either directly or via isomerization of o-Q intermediates suggesting that o-Q is the major reactive toxicophore responsible for reactive metabolite mediated toxicities associated with NDGA use. In addition, we demonstrated that the NDGA-derived dibenzycyclooctadiene lignan (cNDGA 2) undergoes P450-mediated oxidation to a reactive metabolite which might have toxicological implications. There was no evidence of P450-mediated oxidation to reactive metabolites for the phenol-type NDGA analogues. It is concluded that structural modification efforts should focus on phenol-type analogues to potentially enhance the safety profile of NDGA.