The best, direct methods include microscopic investigations. Particularly freeze-fracture electron microscopy have been providing excellent images.

An indirect approach would be quantification of number of phospholipid (PL) molecules per 1, single vesicle.

M. R. Mozafari Thanks for adding answer sir

M. R. Mozafari what protocol do you suggest to do for quantification of number of phospholipid (PL) molecules per 1, single vesicle to differentiate multilamellar from unilamellar vesicles?

Thank you

I suggest you have a look at our paper on the quantification of number of liposomes per volume:

Simple equations pertaining to the particle number and surface area of metallic, polymeric, lipidic and vesicular nanocarriers

MR Mozafari, E Mazaheri, K Dormiani

Scientia Pharmaceutica, 2021, 89 (2), 15

It is an Open Access publication, available online (Free of Charge).