Ribavirin (RBV), a purine analog, causes hemolytic anemia in some patients. In vitro, anemia appears to result from depletion of endogenous purines, but there are limited data in vivo. Single nucleotide polymorphisms in the gene encoding the inosine triphosphatase (ITPA) enzyme have been associated with protection against RBV-induced anemia and may mediate the effect of RBV treatment on endogenous purines. The purpose of this work was to determine the effect of RBV treatment on endogenous purine concentrations in individuals being treated for chronic hepatitis C virus (HCV) infection. Adenosine triphosphate (ATP), guanosine triphosphate (GTP), inosine triphosphate (ITP) and ribavirin triphosphate (RTP) were measured in whole blood obtained from 47 HCV-infected individuals at day zero (baseline), day three, day 28 and day 84 of RBV/sofosbuvir (SOF) treatment. ATP decreased −35.1% and −38.6% (p < 0.0001) at day 28 and day 84 of treatment, respectively compared to baseline. The decrease in ATP was greater in patients with ≤60% ITPA activity compared to those with 100% ITPA activity (−29.4% vs. −9.6%). GTP did not change during treatment but was 16.5% (p = 0.01) higher per 100 pmol/106 cells RTP in those with 100% ITPA activity. No significant change or effect of RTP or ITPA phenotype was noted for ITP. In summary, only ATP was reduced by RBV/SOF treatment and ITPA variants had larger reductions in ATP suggesting RBV-induced anemia is due to a different mechanism than predicted from in-vitro studies. These data emphasize the importance of characterizing the effect of nucleos(t)ide analog treatment on endogenous purines in-vivo.