Determination of the depth of membrane penetration provides important information for studies of membrane protein folding and protein-lipid interactions [1,2]. 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. The knowledge of the immersion depth of fluorescence quenchers (e.g., Doxyl or TEMPO spin probe attached to lipid acyl chains or headgroup) is critical for the determination of the transverse location of fluorescence probes attached to proteins and peptides. The question of bilayer penetration of TEMPO moiety, attached to lipid headgroup in TEMPO-PC, has recently came into prominence in two studies of interfacial solvation [3,4]. Here, we re-examine the arguments on TEMPO penetration using the cross-validation of MD simulations and depth-dependent fluorescence-quenching experiments, which confirms that TEMPO in TEMPO-PC penetrates below the level of phosphate groups. The proper analysis of fluorescence quenching requires the use of Tempo position below the level of phosphate groups; and failure to do so will result in substantial systematic errors in determining the penetration of the labeled site on a membrane protein [5]. Finally, we illustrate the application of this methodology by determining membrane penetration of site-selectively labeled mutants of diphtheria toxin translocation domain [6] and apoptotic inhibitor Bcl-xL [7]. [1] A. S. Ladokhin (2014) Biochim. Biophys. Acta 1838, 2289. [2] A. Kyrychenko et al. (2017) Reviews in Fluorescence 2016: Springer, 243-274. [3] C.-Y. Cheng et al. (2015) Biophys J 109, 330. [4] Y. Lee et al. (2016) Biophys J 111, 2481. [5] A. Kyrychenko et al. (2019) J Membr Biol 252, doi:10.1007/s00232-019-00094-1. [6] A. Kyrychenko et al. (2018) J Membr Biol 251, 379. [7] V. Vasquez-Montes et al. (2019) Biochim Biophys Acta 1867, 691.