The bronopol and kathon are chemical preservative which prevent degradation of milk samples and maintain authenticity in analysis. The detection is based on HPLC–UV–Vis spectroscopy, in which C18 column (250 mm × 4.6 mm, 5 µm) was used for chromatographic separations, with a mobile phase comprising 0.1% phosphoric acid in water: Methanol: 0.1% phosphoric acid in acetonitrile (80:10:10) at a flow rate 0.8 ml/min at ambient temperature and with the UV detection at 250 nm for bronopol and 274 nm for kathon. The retention time of bronopol, kathon (MI 2-methyl-4-isothiazolin-3-one) and kathon (CMI 5-chloro-2-methyl-4-isothiazolin-3-one) was 4.52 min, 3.98 min and 6.68 min respectively with a total run time of 10 min. The linearity of the method was satisfactory with regression coefficient (R2) = 0.99. The limit of quantification was 72, 240, 390 mg L−1 for bronopol, kathon (MI) and kathon (CMI) respectively. Five spiked levels (62.5, 125, 250, 500 and 1000 mg L−1) were used to determine the recovery of bronopol, kathon (MI) and kathon (CMI) which was found to be 95.41 ± 11.84, 95.75 ± 8.21 and 92.22 ± 14.64% respectively, with relative standard deviations in the range 5.9–14.6%. The standardized analytical method was successfully used to rapidly detect bronopol and kathon in milk samples.