Substitution reactions in carbocyanine dyes are used to determine nucleophilic compounds. The interaction of a chlorine-containing carbocyanine dye with a number of medicinal substances is studied. It is shown that, in the reaction with isoniazid in the presence of a surfactant, this dye selectively changes color from yellow-green to violet. The formation of a product of the substitution of chlorine for isoniazid is proven by chromatography–mass spectrometry. The reaction proceeds in 20 min in the presence of 1 mM cetyltrimethylammonium bromide. The limit of detection for isoniazid in water by the photometric method is 10 μg/mL and in diluted artificial urine using fluorimetry, 0.3 μg/mL. The procedure does not use full-spectrum equipment, which simplifies the determination.