Defined key terms: -Antiviral: An agent that kills a virus or that suppresses its ability to replicate and, hence, inhibits its capability to multiply and reproduce. -Method validation: The process of verifying that a method is fit for purpose. -Selectivity: Selectivity is the ability of the bioanalytical method to measure and differentiate the analytes of interest and internal standard in the presence of components which may be expected to be present in the sample. -Precision: The precision of

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... an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained under the stipulated conditions. -Accuracy: The accuracy of an analytical procedure expresses the closeness of the determined value to the value which is accepted either as a conventional true value or an accepted reference value. -Robustness: Robustness is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. Abstract Background: IFN-α5 has demonstrated that induces stronger signaling and higher expression of antiviral genes than IFN-α2, the current treatment in chronic viral hepatitis. However, there is no specific and validated quantification method in order to conduct kinetic studies as part of the preclinical and clinical evaluation for regulatory purposes. Results: A novel integration of an antiviral assay against the cytopathic effect of the encephalomyocarditis virus in HeLa cells with a very sensitive method for assay processing, Vialight ® Plus assay, is presented for IFN-α5 activity quantification. The bioassay has been validated in macaque and human serum and it has demonstrated to be selective, precise and accurate. Conclusion: The validated bioassay meets suitable acceptance criteria for these types of biological assays.