The analysis of pegylated-interferon-alpha(2a) in patient serum samples is of high interest for clinic research trials, as this therapeutic protein has become an important antiviral treatment. In this study, an LC-MS/MS method for the absolute quantification of pegylated-interferon-alpha(2a) in human serum was developed. The assay achieved a lower limit of quantification of 3.6 ng/mL (60 pM) with the use of a monolithic C(18) solid phase extraction to enrich the target protein. The linear range of the assay was defined up to 54 ng/mL to measure the typical clinical pegylated-interferon-alpha(2a) levels, and within this range, the precision and accuracy were found to be within +/-20%. The method was applied to a clinical study and found suitable for high-throughput analysis of pegylated-interferon-alpha(2a) in human serum. In addition, further investigations suggested the enrichment step may have general application to the sensitive analysis of other low molecular weight proteins.