The manuscript deals with the development, validation, and theoretical chemistry behind a reverse-phase HPLC methodology needed to separate, identify, and evaluate the content of 4-nitrophenyl ethylamine (4-NPA) and its associated impurities (i.e., 2-NPA, 3-NPA, 2-PEA, and acetamide impurity). The developed HPLC method was found to be specific, linear, precise, repeatable, accurate, and robust. This method can be used to assess the quality of 4-NPA sample for the quantification of 2-NPA, 3-NPA, 2-PEA, acetamide impurity. The purpose of this research is for separation and quantification of positional isomers, which is mandatory during the analysis of 4-NPA samples to avoid further formation of analogues derivative of 2-NPA and 3-NPA intermediate and drug substances. Computational studies demonstrated that nitro substitution, regardless of its position, enhances the electron-accepting capacity of the molecule and its overall reactivity, while decreasing its stability. The persistent trend of enhanced electrophilicity and electron-accepting characteristics (2-PEA < 2-NPA < 3-NPA < 4-NPA) illustrates the considerable influence of the nitro group position on molecular electronic properties, with para-substitution proving to be the most efficacious in augmenting potential reactivity and electron-accepting capacity.