We report on the quantitative analysis of scanning transmission X-ray microscopy (STXM) images of gas-filled, poly(vinyl alcohol) (PVA)-based microballoons (NIB) in a water environment. A model for the transmitted intensity is proposed on the basis of a perfect spherical shell stabilizing the microballoon. An extension of this model to take into account the deformation of the MBs is also presented. Taking into consideration a density gradient of the shell and the STXM resolution, we were able to explain very precisely two types of experimental STXM profiles observed on gas-filled MBs. This enables the detailed characterization of NIB properties such as radius and wall thickness and the determination of their wall density with unprecedented high resolution.