The application of factorial experimental design is reported in order to evaluate the effect of operating conditions on hydrodesulfurization (HDS) activity evaluated at micro-reaction scale. Reaction temperature, liquid hourly space-velocity (LHSV), amount of catalyst loaded to the reactor, reactor mode of operation (down-flow and up-flow), and catalytic bed dilution with inert material were studied as operating variables. Percentage of sulfur removal was selected as response variable. Experimental results were analyzed by considering main effects of variables and their interactions, as well as analysis of variance, regression analysis, and analysis of residuals. For the case studies, the variables that have greater effect on the HDS activity are the temperature and LHSV, followed by the catalyst amount and the dilution of the catalytic bed. Experimental HDS activity tests of straight-run gasoil (SRGO) were performed in down-flow mode (case study A), up-flow mode (case study B), and up-flow mode with dilution of catalytic bed (case study C). The maximum HDS obtained was 98.30%, 97.49%, and 95.60%, respectively. The regression models developed in this work allows for the prediction of HDS activity of SRGO at certain conditions, with correlation coefficient higher than 0.98 and error less than 0.7%. The proposed methodology can be extended to other situations aiming at defining optimal operating conditions to achieve desired levels of sulfur content in the product.