A new HPLC method using a Polyhydroxyethyl A column involving hydrophilic interaction chromatography (HILIC) is described for the simultaneous determination of urea, allantoin and lysine pyroglutamate in a cosmetic cream. Validation of the method was accomplished with respect to linearity, repeatability and limits of detection/quantification. Compound recoveries approach 100% with acceptable RSD values. The method is very simple since no derivatisation is necessary. Furthermore, it allows the rapid and direct chromatographic analysis of urea and hence could provide an alternative to other methods used to determine this compound in biological or cosmetic samples.