The quantitation of PTH-Fc in circulation by ligand binding assay presented a significant challenge due to the extremely low doses of administration, interference from the endogenous. A robust LC-MS/MS method to quantify the extremely low concentration of PTH-Fc in human serum utilized sequential immunoaffinity enrichment at PTH and Fc domains in conjunction with microflow LC-MS/MS technology significantly improved the sensitivity and selectivity. The assay displayed a quantitation range of 0.025-5.0 ng/ml and acceptable intraday and interday precision (%CV ≤ 15%) and accuracy (%bias ≤ ±15%) and can be routinely used for pharmacokinetic measurement of the drug. The novel sequential immunocapture workflow described herein can be applied to the quantitation of other recombinant therapeutic proteins to support clinical studies.