Imetelstat, a 13-base oligonucleotide (5'-TAGGGTTAGACAA-3'), is a potent, investigational telomerase inhibitor in clinical development for the treatment of hematologic myeloid malignancies. Modifications to imetelstat oligonucleotide chemistry include an N3'-P5' thio-phosphoramidate backbone linkage to improve biologic stability and the addition of a palmitoyl tail at the 5'-position to enhance cellular membrane permeability. Other oligonucleotides have been previously shown to have in vitro test-system-dependent outcomes when potent cytochrome P450 inhibition in human liver microsomes (HLM) is observed, but such inhibition is not observed in cryopreserved human hepatocytes (CHH). Outcomes in CHH are consistent with clinical reports in which no interactions were reported. In the present study, imetelstat was evaluated for in vitro inhibition of eight P450 enzymes, namely CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 in CHH (0.5 million cells/ml). Assays were performed using validated conditions, including short substrate times (10 minutes), and at the approximate substrate Km concentration. Imetelstat was found to have little to no inhibition of all P450 isoforms evaluated, with inhibitor concentration that causes 50% inhibition (IC50) values >100 μM. Maximum percent inhibition values for each P450 isoform at 100 μM imetelstat were <20% except for CYP2C8 activity, which was inhibited by 49%. Using a static mechanistic model, the predicted change in area under the curve of a victim drug coadministered with imetelstat was 1.04-fold, projecting no relevant clinical interaction. Overall, the results from this in vitro study suggest that clinical use of imetelstat is unlikely to affect the pharmacokinetics of concomitant therapies that undergo cytochrome P450-mediated metabolism.