Several techniques are available for making large unilamellar vesicles (LUV) with an average diameter of approximately 100 nm, widely employed as model biomembranes as well as vehicles for drug delivery. Here we describe the use of adaptive focused ultrasound (AFU) for the preparation of LUV from multilamellar vesicles (MLV) and studied the effects of ultrasound intensity and number of cycles per burst (CPB) on the average size of vesicles produced. CPB determines the duration of the intermittent pressure wavetrains emitted by the transducer, and the corresponding relaxation periods. Preliminary experiments indicated that CPB controls the size of vesicles assembling after the disruption of MLV by ultrasound and optimum values for obtaining LUV could be iterated. The sizes and lamellarity of LUV were assessed by dynamic light scattering (DLS), cryogenic transmission electron microscopy (cryo-TEM), and fluorescence quenching. AFU provides a simple and easy to use approach for making liposomes with several advantages: it is minimally invasive and involves no loss of material. Precisely controlled wavelengths are employed with a significant reduction in the presence of hot spots, which could destroy some biological materials of interest.