For targeted therapies, immunocapture-liquid chromatography mass spectrometry (IC-LC/MS) technology has become an important tool for determination of both drug exposures, target antigen densities, and engagement in the systemic circulation and/or in total target tissue homogenates. Although the information collected from the circulation and tissue homogenates is useful in establishing the correlations of the exposure and target engagement with the pharmacodynamic response and efficacy of a therapy, the measurement at the cell plasma membrane within the target tissue is preferred, since it is the primary action site for antigen and the target drug. However, to the best of our knowledge, IC-LC/MS-based methodologies to conduct the assays at the plasma membrane from tissue sample has been quite limited. In this paper, we reported an IC-LC/MS-based assay platform for assessing the target engagement in tumor plasma membrane prepared from the tumor tissue samples. In addition, tumor samples with guanylyl cyclase C (GCC) expression after fully human IgG1 monoclonal antibody-based antibody-drug conjugate (ADC) treatment were used as a case study. The methodology can differentiate between the total and target-drug bound fraction of GCC with minimal potential equilibrium shift between in-cell surface protein and organelle protein in tumor samples to calculate in vivo target engagement. This approach to determine in vivo target engagement in tumor plasma membrane will provide better understanding of pharmacokinetic/pharmacodynamic relationship to achieve the desired antitumor efficacy.