Equine infectious anemia virus (EIAV) infection often results in an initial febrile response, followed by recurrent cycles of the disease and finally, a prolonged asymptomatic period. These variations in clinical signs are due to a number of factors, including virus strain, equid species and differences in susceptibility among animals. As a consequence of the close relation between viral strain and disease, studies about in-vitro replication and fitness of EIAV in macrophages, which are the target cells of the virus, depend on accurate measurement of viral load throughout the infection period. We developed a method to quantify EIAV in-vitro using a one-step RT-qPCR system from a control RNA synthesized for this purpose. Designed primers amplified a 520 base pair fragment from the gag gene region that was inserted into a pGEM-T Easy Vector plasmid and propagated in Escherichia coli DH5-α.The bacteria with the construct were propagated and sufficient quantities of the template DNA were produced. The RNA was synthesized in-vitro from the plasmid linearization product and was used for standardization of a one-step RT-qPCR system with a minimum detection limit of 10 to 15 molecules. The efficiency of the reaction was 101%, with r2 equal to 0.997. This new method can be used for the determination of virus titer in EIAV replication studies in-vitro.