The compound 2,6-di-tert-butyl-4-nitrophenol (DBNP), a potentially powerful uncoupler of ATP-generating oxidative phosphorylation, has been physically and spectroscopically characterized using X-ray crystallography, differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), GC-MS spectrometry, Fourier-transformed IR (FTIR) spectrophotometry, UV-Vis spectrophotometry, and FT 1H- and 13C-NMR spectroscopy. However, DBNP is not commercially available; therefore, it had to be synthesized in the laboratory prior to toxicity studies. The DBNP was prepared from 2,6-di-tert-butylphenol (DBP) precursor in hexane through an electrophilic aromatic substitution process using NO2. A collective yield of 75% was obtained by using two empirically determined end points that prevented the coprecipitation of reaction by-products and resulted in the formation of DBNP in high purity. Excessive amounts of NO2 in reaction mixtures resulted in the decomposition of preformed DBNP. With a pKa value of 6.8 and a higher degree of lipophilicity, DBNP may prove to be a stronger uncoupler of oxidative phosphorylation than 2,4-dinitrophenol (pKa = 4.09) due to the expected enhancement of passive-diffusion kinetics across biological membranes at the physiological pH of 7.4. The present study is intended to provide analytical toxicologists, industrial hygiene monitors, and other professionals involved in chemical health and safety with a comprehensive source of basic information on the synthesis and analytical chemistry of DBNP.