As a result of their improved aqueous solubility, the development of phosphate ester prodrugs is an interesting approach to increase intestinal absorption of poorly water-soluble drugs. Absorption of a drug from its phosphate ester prodrug is based on intestinal dephosphorylation of the prodrug which may result in intraluminal supersaturation of the parent drug, followed by an increased absorptive flux across the intestinal mucosa. In this study, we evaluated the behavior of fosamprenavir, a phosphate ester prodrug of amprenavir, in the Caco-2 system and in aspirated human intestinal fluids (HIF), both showing phosphatase activity. Starting from a solution of fosamprenavir in HIF, a supersaturated solution of amprenavir was generated and maintained during a time period sufficient for absorption. Moreover, supersaturation of amprenavir resulted in an enhanced flux across Caco-2 monolayers. To our knowledge, this is the first illustration of supersaturation in real intestinal media. Next, we showed an inhibitory effect of inorganic phosphate on the dephosphorylation of fosamprenavir, both in the Caco-2 model and in HIF. As a consequence, phosphate-buffered media, including fasted state simulated intestinal fluid (FaSSIF), are incompatible with the study of phosphate ester prodrugs and should be replaced with media containing a biorelevant phosphate concentration (0.4-1 mM) and another buffering compound such as 2-morpholinoethanesulfonic acid (MES).