Abstract A new separation method based on a novel reversed-phase sequential injection chromatography (SIC) technique was used for simultaneous determination of ambroxol hydrochloride and doxycycline in pharmaceutical preparations in this contribution. The coupling of short monolith with SIA system results in an implementation of separation step to until no-separation low-pressure method. A Chromolith ® Flash RP-18e, 25–4.6 mm column (Merck, Germany) and a FIAlab ® 3000 system (USA) with a six-port selection valve and 5 ml syringe were used for sequential injection chromatographic separations in our study. The mobile phase used was acetonitrile–water (20:90, v/v), pH 2.5 adjusted with 98% phosphoric acid, flow rate 0.48 ml min −1, UV detection was at 213 nm. The validation parameters have shown good results: linearity of determination for both compounds including internal standard (ethylparaben) >0.999; repeatability of determination (R.S.D.) in the range 0.5–5.4% at three different concentration levels, detection limits in the range 0.5–2.0 μg ml −1, and recovery from the pharmaceutical preparation in the range 99.3–99.9%. The chromatographic resolution between peak compounds was >5.0 and analysis time was <9 min under the optimal conditions. The method was found to be applicable for routine analysis of the active compounds ambroxol hydrochloride and doxycycline in various pharmaceutical preparations.