Obtaining a proper bile sample for investigative purposes is of utmost importance to obtain valid results. Bile can be collected by direct aspiration of the gallbladder, by duodenal intubation or by T-tube drainage. The optimal method of collection depends on the investigative question, as well as on the resources available to the investigator. The procedures for obtaining, processing and analyzing human bile (gallbladder and hepatic) are summarized, pointing out the disadvantages and pitfalls that may occur.