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