This paper deals with searching of HPLC chromatographic conditions for determination and separation of Transkarbam 12 (T 12) and its two main degradation products (omega-aminocaproic acid and dodecylalcohol). T 12 is a new substance which belongs to the group of accelerators of transdermal penetration. Chromatographic separation was achieved using Separon SGX C18 analytical column (150 mm x 3 mm i.d.; 5 microm). Mobile phase contained acetonitrile and sodium acetate buffer pH 4.5 at the flow rate of 1 ml/min. Separation was carried out under the conditions of gradient elution. After the modification of the structure by derivatization reagent (3,5-dinitrobenzoyl chloride) detection at wavelength 230 nm was realized. The aim of this study was not only the optimization of the separation of derivatization reagent and derivatized T 12, Ak and D but also optimal derivatization processes for all three substances.