||Determination of the depth of membrane penetration provides important information for studies of membrane protein folding and protein-lipid interactions. Here, we use a combination of molecular dynamics (MD) simulations and depth-dependent fluorescence quenching to calibrate the methodology for extracting quantitative information on membrane penetration. In order to investigate the immersion depth of the fluorescent label in lipid bilayer, we studied 7-nitrobenz-2-oxa-1,3-diazole (NBD) attached to the lipid headgroup in NBD-PE incorporated into POPC bilayer. The immersion depth of NBD was estimated by measuring steady-state and time-resolved fluorescence quenching with spin-labeled lipids co-incorporated into lipid vesicles. Six different spin-labeled lipids were utilized: one with headgroup-attached Tempo probe (Tempo-PC) and five with acyl chain-labeled n-Doxyl moieties (n-Doxyl-PC where n is a chain labeling position equal to 5, 7, 10, 12, and 14, respectively). The Stern-Volmer analysis revealed that NBD quenching in membranes occurs by both static and dynamic collisional quenching processes. Using the methodology of Distribution Analysis, the immersion depth and the apparent half-width of the transversal distributions of the NBD moiety were estimated to be 14.7 and 6.7 Å, respectively, from the bilayer center. This position is independently validated by atomistic MD simulations of NBD-PE lipids in a POPC bilayer (14.4 Å). In addition, we demonstrate that MD simulations of the transverse overlap integrals between dye and quencher distributions can be used for proper analysis of the depth-dependent quenching profile. Finally, we illustrate the application of this methodology by determining membrane penetration of site selectively labeled mutants of diphtheria toxin T-domain.