Surrogate planar and membrane systems have been employed to study the architecture of immune synapses; however, they often do not recapitulate trans-synaptic extraction and endocytosis of ligands by the immune cells. Transendocytosis (or trogocytosis) of antigen from immune synapses is particularly critical for antigen processing and presentation by B cells. Here we describe a protocol for preparation of plasma membrane sheets (PMSs), which are flexible and fluid membrane substrates that support robust B cell antigen extraction. We show how to attach B cell antigens to the PMSs and how to investigate antigen extraction and endocytosis by fluorescent microscopy and computational image analysis. These techniques should be broadly applicable to studies of transendocytosis in a variety of cellular systems.