CYP20A1 is a well-conserved member of the human cytochrome P450 enzyme family for which no endogenous or xenobiotic substrate is known. We have recently shown that this enzyme has moderate activity towards two proluciferin probe substrates. In order to facilitate the search for physiological substrates we have tested nine additional proluciferins in this study and identified three such probe substrates that give much higher product yields. Using one of these probes, we demonstrate inhibition of CYP20A1 activity by 1-benzylimidazole, ketoconazole and letrozole. Finally, we show that the combination of two common single nucleotide polymorphisms (SNPs) of CYP20A1 leads to an enzyme (CYP20A1Leu97Phe346) with reduced activity.