Context <p>The presence of potentially carcinogenic nitrosamines in drugs has been a worldwide concern, driving strategies to control or mitigate their formation to protect patient health. Understanding the critical factors for <i>N</i>-nitrosation, such as mechanisms and energy barriers, enhances the risk assessment process to understand potential nitrosamine formation. Evaluation of the structural impact of amines on the <i>N</i>-nitrosation rate in the presence of nitrites and acidic media is of great interest to pharmaceutical companies assessing the risk of nitrosamine drug substance–related impurities. A range of secondary amines was explored using DFT calculations to assess the impact of electronic and steric effects on activation energy. <i>Asym</i>-N₂O₃ was selected as the nitrosating agent since its reaction was shown to be favorable following screening of pathways employing nitrosyl chloride, nitrous acid, <i>asym</i>-N₂O₃, <i>sym</i>-N₂O₃, and <i>trans-cis</i>-N₂O₃. The relatively low activation energies obtained for all amines indicate the reaction is very likely to occur if the reactive components encounter, even for amines with sterically hindered and electron-withdrawing groups. Understanding the interaction between the amine and nitrosating agent is therefore the defining factor in the risk of formation of more complex nitrosamines within drugs.</p> Methods <p>Calculations were performed using the Gaussian-16 program. The B3LYP-D3/def2-TZVP level of theory was applied for structure optimizations. The IEF-PCM implicit model was used for the solvent effect. Intrinsic reaction coordinate calculations were carried out to connect the transition state with the associated minimum.</p> Graphical Abstract <p></p>