Ribavirin, a nucleoside analog, is administered in combination with interferon to patients with chronic hepatitis C. To evaluate the feasibility of ribavirin therapeutic drug monitoring, we investigated the influence of blood collection and preanalytical conditions on ribavirin concentrations and compared the results obtained from interlaboratory blind tests by 3 laboratories using different analytical techniques. On 3 occasions, blank serum samples spiked with ribavirin and pooled serum samples from patients on ribavirin were sent to the 3 laboratories. Two analytical techniques were based on liquid chromatography with tandem mass spectrometry (LC-MS/MS) and 1 on high-performance liquid chromatography with UV detection (HPLC-UV), with protein precipitation or solid-phase extraction, all validated according to international guidelines. Inter- and intra-batch mean relative errors ranged from −7.4% to +10.3% and from −10.3% to +7.4%, respectively. Relative standard deviations were <13.5% and <10.6%, respectively. Linearity, assessed blindly, between 125 and 4550 ng/mL was excellent (r > 0.991) for all 3 methods. The 2 LC-MS/MS techniques were slightly less precise and accurate than HPLC-UV, perhaps because the internal standard used was not a ribavirin isotope. Accurate and precise LC-MS/MS and HPLC-UV methods developed in 3 different laboratories provided excellent and consistent results to blind tests for ribavirin determination in spiked serum samples and pools of serum samples from patients with chronic hepatitis C virus infection.