A straightforward methodology, based on first principles, for the estimation of human cytochrome P450-substrate binding energies is outlined, and the system has then been applied successfully to a relatively large dataset of P450 substrates totalling 90 compounds. The results of Quantitative Structure-Activity Relationship (QSAR) analysis on the same dataset of cytochrome P450 (CYP) substrates from the CYP1 , CYP2, and CYP3 families, involving a total of 90 compounds, agree favourably with the original analysis based on first principles, thus confirming the use of average values for hydrogen bond and π-π stacking energies, together with utilizing log P values as an estimation of desolvation energies. This method is based on a linear summation of the various contributary factors to the process, including: desolvation, hydrogen bonding, π-π stacking, restricted bond rotation and other energies relating to loss in translational and rotational energy. It is found that, for the majority of P450 substrates investigated, the first four terms are required for a relatively good estimation (R = 0.98) of the substrate binding affinity (ΔGbind) towards CYP1 and CYP2 enzymes. Consequently, it would appear that the loss in rotational and translational energy, which is thought to occur on substrate binding, apparently has little effect in most cases, possibly due to some degree of residual motion of the enzyme-substrate complex within the endoplasmic reticulum membrane. However, the appearance of a small constant term in the QSAR equation could possibly relate to an average loss in translational and rotational energy for the 90 compounds studied in this investigation.