Use of biosurfactants in bioremediation, facilitates and accelerates microbial degradation of hydrocarbons. CTAB/MB agar method created by Siegmund & Wagner for screening of rhamnolipids (RL) producing strains, has been widely used but has not improved significantly for more than 20 years. To optimize the technique as a quantitative method, CTAB/MB agar plates were made and different variables were tested, like incubation time, cooling, CTAB concentration, methylene blue presence, wells diameter and inocula volume. Furthermore, a new method for RL detection within halos was developed: precipitation of RL with HCl, allows the formation a new halos pattern, easier to observe and to measure. This research reaffirm that this method is not totally suitable for a fine quantitative analysis, because of the difficulty to accurately correlate RL concentration and the area of the halos. RL diffusion does not seem to have a simple behavior and there are a lot of factors that affect RL migration rate.