This work describes atomic force microscopy studies of the physical parameters determining the strength oflipid anchorage in bilayers as a function of the phase state of the lipid matrix. These investigations usedbiotinylated lipids and streptavidin-derivatized tips to quantify the lipid pullout force from different lipidmatrices. Analysis of the data using both dynamic force spectroscopy and full microscopic models show thatthe anchorage strength is greater in gel-phase relative to fluid-phase lipids. Additional model parameter estimatesprovide further insights into the hidden energy barriers that determine the mechanical integrity of lipid anchorsin biological membranes.