Anticoagulants are used to prevent clot formation both in vitro and in vivo. In the specific field of in vitro diagnostics, anticoagulants are commonly added to collection tubes either to maintain blood in the fluid state for hematological testing or to obtain suitable plasma for coagulation and clinical chemistry analyses. Unfortunately, no universal anticoagulant that could be used for evaluation of several laboratory parameters in a sample from a single test tube is available so far. Ethylenediamine tetraacetic acid (EDTA) is a polyprotic acid containing four carboxylic acid groups and two amine groups with lone-pair electrons that chelate calcium and several other metal ions. Calcium is necessary for a wide range of enzyme reactions of the coagulation cascade and its removal irreversibly prevents blood clotting within the collection tube. Historically, EDTA has been recommended as the anticoagulant of choice for hematological testing because it allows the best preservation of cellular components and morphology of blood cells. The remarkable expansion in laboratory test volume and complexity over recent decades has amplified the potential spectrum of applications for this anticoagulant, which can be used to stabilize blood for a variety of traditional and innovative tests. Specific data on the behavior of EDTA as an anticoagulant in hematology, including possible pitfalls, are presented. The use of EDTA for measuring cytokines, protein and peptides, and cardiac markers is described, with an outline of the protection of labile molecules provided by this anticoagulant. The use of EDTA in proteomics and in general clinical chemistry is also described in comparison with other anticoagulants and with serum samples. Finally, the possible uses of alternative anticoagulants instead of EDTA and the potential use of a universal anticoagulant are illustrated.