This study presents TLC, HPLC-DAD and HPLC-FT-IR analyses concerning the detection, identification and determination of organic impurities of commercial tropicamide ((R,S)-N-ethyl-3-hydroxy-2-phenyl-N-(4-pyridylmethyl)propanamide) as a medical substance designed for the production of eye drops. In the examined samples from several random production batches, only one impurity (defined by Ph. Eur. 6th Ed.) was discovered in the amount sufficient for the quantitative analysis. On the basis of comparison of retention times, UV and IR spectra of the impurity and its synthesized standard, this impurity was identified as apotropicamide (N-ethyl-2-phenyl-N-(4-pyridylmethyl)prop-2-enamide). For the chemical identification of organic compounds occurring in the tropicamide samples, an off-line coupling of HPLC with FT-IR was used. The structure of a standard of apotropicamide was confirmed by (1)H NMR and (13)C NMR analysis. Validation of the HPLC-DAD method of determination both tropicamide and apotropicamide in the tropicamide medical substance was performed. This method is suitable for use in the quality control of tropicamide during its production.