This work investigates the use of positron annihilation lifetime spectroscopy (PALS) for the in-situ structural characterisation of silica derived thin film membranes. By using a quantified maximum entropy method, PALS allowed for the measurement of a pore size distribution depth profile. PALS measurements were carried out on a series of silica derived membranes where alumina supports were coated with four layers of cationic HTBA surfactant cobalt silica sols wherein the surfactant/cobalt molar ratio loading varied from 0 to 3. PALS results showed that the coated layers adjacent to the porous alumina substrate were characterised by micropores and broad mesopores, a clear indication that the porosity of the substrate affected the pore size at the substrate and thin film interface. The last coated layer resulted in a high concentration of ultra-micropores (dp < 6 Å). This was attributed to the surface smoothness conferred by three previous coated layers. Higher surfactant loadings resulted in an increase in gas permeation and reduction of He/CO2 permselectivity from 91.5 to 3.8. A strong correlation with R2 up to 0.999 was found between the ultra-micropores in the top layer and gas permselectivity, a clear indication that gas separation is controlled by small pore sizes. In this work, PALS showed to be a powerful tool for the characterisation of the structural features of thin films.