This protocol presents a variation on the 2^-ΔΔCt technique for qPCR analysis. Our approach requires the inclusion of a standard curve on each qPCR plate, and like the 2^-ΔΔCt technique, is dependent on the stability of housekeeping gene expression. However, unlike the 2^-ΔΔCt technique, our approach corrects for imperfect cDNA amplification efficiency and allows for the use of multiple housekeeping genes. Collectively, this approach enhances analytical accuracy and thereby reduces the type I and II statistical errors in the generated data.