Three new degradants have been identified from drug product and active pharmaceutical ingredient stability samples of aztreonam, a marketed synthetic monocyclic beta-lactam antibiotic. The degradants were detected following the implementation of a new, more selective HPLC method for the determination of impurities and degradants. The new method was developed in response to changes in the regulatory requirement for mature products. Two of the new unknown Degradants (I and II) were observed in chromatograms from stability samples of aztreonam injection. The third new Degradant (III) was observed during a stability study of the aztreonam active pharmaceutical ingredient. These degradants were structurally characterized. A small amount (ca. 1–3 mg) of each degradant was isolated via preparative HPLC for structure elucidation using accurate MS, one and two-dimensional NMR spectroscopy. The small amount of each NMR sample was then reused as a standard for HPLC purity/impurity method validation. Their exact concentrations were determined using quantitative NMR which enabled the execution of the quantitative elements of the HPLC method validation. This innovative approach eliminated the need to isolate or synthesize larger quantities of markers for HPLC/UV method validation, thus saving significant time and reducing costs.