A HPLC method was developed to quantify rhamnolipid species in a bacterial biosurfactant mixture. The biosurfactant mixtures containing mainly 3-[3'-(L-rhamnopyranosyl-oxy)decanoyloxy]decanoic acid (RhC10C10), 3-[3'-(2'-O-alpha-L-rhamnopyranosyl-oxy)decanoyloxy]decanoic acid (Rh2C10C10),