The monitoring and manipulating of the behaviors of natural or artificial materials on lipid vesicles have become an important research topic because it provides basis for elucidating cell functioning and designing vesicle drug delivery system. Here, we report the explicit understanding of the multistep cross-membrane kinetic of charged dyes and an anticancer drug during the interacting with single-component unilamellar lipid vesicles. Comparing the outside-to-inside and inside-to-outside translocation and studying the effect of ionic strength, we obtained details in these dynamic processes using second-harmonic generation technique. Experimental results demonstrated that the rod-like D289 (4-(4-diethylaminostyry)-1-methyl-pyridinium iodide), the planar malachite green chloride dyes, and the L-shaped anticancer drug doxorubicin hydrochloride molecules crossed 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt lipid bilayer mainly in the charged form by passive diffusion. Besides, the permeability of these charged molecules across lipid bilayer can be notably facilitated by the increased ionic strength induced by millimole of NaCl in the outside environment of the vesicles. This effect can be interpreted with the electrostatic screening effect using a quantitative model based on the transmembrane electrochemical potential. This work provided a clear understanding on the dynamics and migration forms for the transmembrane delivery of charged molecules and a simple strategy for regulating these dynamic processes.