Hookworm is generally caused by Necator americanus (Stiles, 1902), causing digestive symptoms and anemia. The diagnosis by coprology can have low sensitivity, especially in light parasite loads. The Polymerase Chain Reaction (PCR) is a sensitive and specific technique, which must be adapted to laboratory conditions and positive controls are necessary. The objective of this work was the standardization of the PCR technique for the amplification of the ITS-1 sequence of N. americanus in stool samples and its cloning for its use as a control. Three DNA extraction protocols were standardized (Phenol/chloroform, Saline Precipitation, and Chelex® 100 Resin). Optimal reagent concentrations (MgCl , BSA, dNTP, primers, and Taq polymerase) were determined, as well as the hybridization 2 temperature and number of cycles. The analytical sensitivity and specificity of the technique was determined. F...