The influence of the main chromatographic parameters on the conduct of the HPLC-UV assays was elaborated on the elution of four vitamers (retinol, retinyl acetate, tocopherol and tocopheryl acetate). Considering, both type A and type B methods to estimate their measurement uncertainty magnitudes, taking into account the specifications information and the repeated available data. In this context, the retention time variability was used to cover the effect of the most factors. Likewise, the areas data for all peaks of interest were randomly generated on current chromatograms by the software, to examine the baseline effect on the peaks integration. As a result, the peak area's variability, which reaches up to 5%, indicates that the uncertainty contribution, in area measurement is mainly due to the baseline effect, which make difficult the peaks integration, especially for low amount compounds. Nevertheless, the low variability between runs in the retention time reflects much more the stability of the HPLC-UV system and attests that the influences of the other chromatographic parameters remain insignificant.