The quantitative determination of tacrolimus was studied by using reversed-phase high performance liquid chromatography (RP-HPLC). Due to tacrolimus two kinds of conformational heterogeneity a series of mobile phases were tested, in order to achieve the most suitable one. Each test was performed in a C18 column at 20, 40, and 60 °C. An increasing in temperature and acetonitrile content on mobile phase led to a suitable system to quantify tacrolimus in fermentation broth.