Liquid chromatography tandem mass spectrometry (LC-MS/MS) has been shown to be a viable tool for preclinical pharmacokinetic (PK) analysis of monoclonal antibody (mAb) therapeutics. This work describes free and total PK assays for the mAb PF-00547,659 in serum of ulcerative colitis patients in a First-In-Human study [Vermeire, S. et al. Gut2011, 60 (8), 1068-1075]. The assay to measure free PF-00547,659 used immuno-enrichment with a biotinylated anti-idiotypic antibody and streptavidin magnetic beads. The total assay used enrichment by protein G magnetic beads. Following elution of PF-00547,659 from the beads, addition of an extended sequence stable isotope labeled peptide and trypsin digestion, a proteotypic peptide derived from the CDR region of the light chain of PF-00547,659 was quantified by LC-MS/MS. The free assay had a calibration range from 7.03 ng/mL to 450 ng/mL. The assay was precise and accurate with interbatch imprecision <16.5%, and interbatch inaccuracy <13.7% at all concentrations investigated during assay qualification. Results from LC-MS/MS methodologies are compared with historical immunoassay data originally acquired during the course of the clinical study. PK parameter estimates were highly correlated between the two analytical approaches. This work provides precedence that immunoaffinity LC-MS/MS can effectively be used to measure the serum concentrations of mAb therapeutics in clinical studies.