Nuclear magnetic resonance (NMR) technique has been used to find the property of atropisomerism in Rilpivirine hydrochloride by variable temperature analysis and various 2D techniques. Both the Rilpivirine hydrochloride (E-isomer) and Impurity-A (Z-isomer) isomers have been differentiated and confirmed by NMR and ultraviolet techniques. Preparative high-performance liquid chromatography isolation for Impurity-A is followed by spectroscopic (NMR, mass spectra, and infrared) investigation that provides a perfect solution for determination of the structure of Rilpivirine and related impurities.