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Steady state kinetic assays have been a reliable way to estimate fidelity of several polymerases (Menendez-Arias, 2009; Rezende and Prasad, 2004; Svarovskaia et al., 2003). The ability to analyze the extension of primers with specific mismatches at the 3ʹ end is a major strength of the mismatched primer extension assays. Recently, we used the mismatched primer extension assays to show that the fidelity of HIV RT increases dramatically when concentration of Mg2+ is reduced to a physiologically relevant concentration (~0.25 mM) (Achuthan et al., 2014). Here, we describe in detail how to perform the mismatched primer extension assay to measure the standard extension efficiency using human immunodeficiency virus reverse transcriptase (HIV RT) at 2 mM Mg2+. The relative fidelity of the polymerase can then be estimated using the standard extension efficiency. The assay described here is based on the method published in Mendelman et al. (1990).