Rhamnolipids, produced by Pseudomonas aeruginosa, represent an important group of biosurfactants having various industrial, environmental, and medical applications. Rhamnolipids are expensive to produce due to the highly foaming nature and complex metabolic regulations involved. Considerable efforts have been directed in the past years to reduce the production costs by improving the yield, studying genetically engineered strains, and using either low cost feedstock or agricultural byproducts as substrates. A fast method for rhamnolipid analysis can significantly enhance the strain selection, metabolic engineering, and process development for improved production.  The currently used methods are tedious and laborious.  A qualitative method was proposed earlier to differentiate rhamnolipid producing and non-producing strains using agar plates containing methylene blue and cetyl trimethylammonium bromide (CTAB).  By systematically investigating the complexation of rhamnolipids and methylene blue, with and without the presence of CTAB, we have developed a rapid and simple method for rhamnolipid analysis.  It relies on measuring the absorbance (at 638 nm) of the rhamnolipid-methylene blue complex that partitions into the chloroform phase. This method has been verified with the results obtained from the commonly used anthrone reaction technique