Microbial-produced biosurfactant glycolipids consist of four major groups: rhamnolipids, sophorolipids, trehalose lipids and mannosylerythritol lipids. Extensive research has been carried out on the production and analysis of rhamnolipids and sophorolipids, with significantly less research publications in the area of trehalose lipids and mannosylerythritol lipids. When studying these microbial glycolipids, the ability to isolate, purify and characterise the structures produced by fermentation process is extremely important. Chemical and structure information obtained from the glycolipid products provides invaluable information on the different conditions, such as microbial strain type, carbon sources and temperature, which influence production of glycolipids. The information from quantification and chemical analysis of these glycolipids assists in the optimisation of production yields and assembly of glycolipids with different structural characteristics. Armed with this information, the manufacture of glycolipids can be manipulated with different fermentation approaches to tailor the production of different types of glycolipid depending on the biological activity required. The experimental techniques used to isolate, purify and analyse glycolipids are extremely varied with each method having a range of pros and cons. These methods range from simplistic methods such as colorimetric assays that provide rough indication of production yields in an inexpensive manner, extending to complex mass spectral techniques, which necessitate sophisticated instrumentation and scientific expertise. This chapter therefore details information and protocols that are essential for the detection and chemical characterisation of glycolipids. Since there is an abundance of methods available, only the most commonly described techniques are detailed in this chapter.