A sensitive method for determination of PEG-IFN-α-2b in human serum was developed using ultra performance liquid chromatography aligned with tandem mass spectrometric detection. A two-treatment, two-period, cross over study was conducted to establish bioequivalence between a test and reference formulation and the method was successfully applied to the quantification of PEG-IFN-α-2b in serum samples of this clinical study. The sample concentrations obtained from LC-MS/MS technique were compared with the concentrations obtained from ELISA technique. PEG-IFN-α-2b was isolated from serum using protein precipitation technique with isopropyl alcohol followed by overnight tryptic digestion. The signature peptide formed as result of tryptic digestion was separated on a chromatograph and detected using a mass detector. The mass transition ion-pair of m/z 741.3 → 1047.1 for PEG-IFN-α-2b and m/z 387.4 → 205.2 for internal standard were used for MS/MS detection. The sample extraction involves a simple protein precipitation method followed by tryptic digestion of the supernatant and further sample cleanup was not needed. The method has been validated over a linear range of 1.028-3200 ng/mL with a correlation coefficient ≥ 0.99. The precision (%RSD) was 5.52 to 7.90 and accuracy (%RE) was within -1.80 to 1.68. The total run time was 22.0 min. The sensitivity of LC-MS/MS method was 1.0 ng/ml which was found to be more sensitive than ELISA and resulted in improving the overall study data by being able to quantify all the samples without any below LOQ results helping to further improve the pharmacokinetic modeling. This improved method is a promising anti-body free LC-MS/MS based methodology for estimation of PEG-IFN-α-2b in human serum and may be applied for other such pegylated molecules.