This presented paper deals with a methodology for the separation and simultaneous determination of two active substances in topical pharmaceutical formulation composed of lidocaine (L) and prilocaine (P). The methodology described is based on the sequential injection chromatography (SIC) with UV detection. Monolithic Column Chromolith Flash RP-18, 25mmx4.6mm (Merck, Germany) was used. Separation was performed using elution with binary mobile phase composed of acetonitrile-phosphate buffer 0.05M (40:80 (v/v))+0.01% triethylamine (adjusted to pH 7.1 with H(3)PO(4)) at a flow rate of 0.6mlmin(-1). The analysis duration was <7min. The method was linear over the range of 2.5-200mgl(-1) with a detection limit of 0.25mgl(-1) for both substances. The system was then coupled with Franz cell. Fully automated system for the in vitro release testing of semisolid dosage forms based on sequential injection analysis (SIA) was developed. Simultaneous measurement of L and P release was done by this system. Samples were taken in 10.5min intervals during 4h of the release test. Each test was followed by calibration with five standard solutions. Receiving medium was replenished automatically by the system.