“Stealth” liposomes containing poly(ethylene glycol) (PEG) are good candidates for drug delivery due to their biocompatibility. Additionally, interest in using ultrasound energy in intracellular drug delivery has been growing rapidly. Combining the technology of ultrasound and the science of lipids will lead to advancements in the field of targeted drug delivery. Development and treatment optimization of such non-diagnostic applications of ultrasound requires a fundamental understanding of interactions between the acoustic wave and phospholipid membranes, be they cell membranes or liposome bilayers. This work investigates the changes in membrane permeation (leakage mimicking drug release) in vitro during exposure to ultrasound applied in two frequency ranges: “conventional” (1 MHz and 1.6 MHz) therapeutic ultrasound range and low (20 kHz) frequency range. Phospholipids vesicles were used as controllable biological membrane models. Membrane properties were modified by changes in vesicle dimensions and incorporation of PEGylated lipids. The results of this work might be helpful to optimize acoustic field and membrane parameters for gene or drug delivery.