Biological membranes have two leaflets that can differ in both lipid composition and total lipid abundance. These different types of asymmetries play a major role in determining the biophysical properties of the membrane; however, they have proven challenging to assay experimentally even in simpler model systems. Molecular dynamics simulations offer the means for detailed computational investigation of systematically varied interleaflet lipid distributions, but opportunities for critical validation with wet lab experiments are scarce. To help address this problem, here we use atomistic simulations of asymmetric bilayers to generate synthetic experimental data and thus investigate the sensitivity of various approaches to changes in relative lipid composition, number, and cholesterol distribution. Contrary to trends in symmetric bilayers, the simulations showed a decrease in lipid packing with increasing cholesterol in differentially stressed asymmetric bilayers, with more pronounced changes in the more loosely packed leaflet. Representative experimental data computed from the simulation trajectories indicated that the detection of asymmetry-induced changes in leaflet properties should be possible with environment-sensitive fluorescent probes and NMR observables, but may require optimization of sample preparation conditions. On the other hand, small-angle scattering data are already experimentally accessible and can reveal differential leaflet packing densities through a model-free analysis. We further show that computationally generated cryo-EM intensity profiles are highly sensitive to phospholipid imbalance between membrane leaflets. Together, these findings provide a roadmap for developing targeted applications of the in vitro techniques and obtaining experimental data critical for validating computationally derived principles related to membrane asymmetry.