1. Resuspend RNA and DNA oligos to 500 µM in 100 mM Tris-HCl (pH 8.1), 0.5 mM EDTA (pH 8.0). 2. Prepare annealing solution of 50 µM RNA/DNA oligos with 50 mM NaCl in DNase/RNase-free water, aliquot 50 µl in PCR tube, vortex and spin down briefly. 3. Place tube in thermal cycler: heat to 95 °C for 2 min, ramp cool to 25 °C over 45 min, store at 4 °C. 4. Store RNA/DNA hybrid solution at -20 °C. 5. Dissolve inhibitor compound in DMSO to 10 mM, sonicate and warm if needed, prepare serial dilutions in Milli-Q water. 6. Plan experiment with blank (no RT enzyme), control (RT enzyme, no inhibitors), and samples (RT enzyme with increasing inhibitor concentrations). 7. Add water (20 µl in blanks, 10 µl in controls) to 96-well plate. 8. Add 80 µl RT reaction mix (1.25x). 9. Add 10 µl inhibitor dilution to samples, 10 µl enzyme (20 ng HIV-1 RT or 2 nM PFV RT) to each well. 10. Incubate plate at 37 °C for 1 h at 200 rpm. 11. Stop reaction with 50 µl EDTA (0.5 M, pH 8.0). 12. Quantify reaction with Victor 3 at 490/528 nm, subtract blank value from samples, report inhibitor values as percentage of control. 13. Calculate IC50 value as the concentration reporting 50% reduction of signal compared to control.