Validation of analytical methods for poorly soluble BCS Class II/IV drugs like Aprepitant in aqueous media is complicated due to instability of samples at the stage of preparation. The goal of the research is to detect the mechanism of loss of Aprepitant analytical signal during filtration and determine the conditions for an exact diagnosis. In this paper, the effect of membrane materials (PES, PVDF, Nylon, PP) on analyte concentration measurements via UV spectrophotometry was examined. Filtration of aqueous solutions is established to cause significant analyte losses ranging from 15.1% (PP) to 83.9% (PES), depending on the initial concentration. It is proved that the main loss factor is not passive adsorption, but mechanically induced heterogeneous nucleation on the membrane surface, which is confirmed by an increase in optical scattering within the 300–500 nm range. Fraction filtration revealed abnormal desorption effects on PES filters. It has been shown that the use of an acidic medium (0.1 M HCl) prevents nucleation due to protonation of the molecule: filtration losses are reduced to statistically insignificant values (<1.6%). To ensure the accuracy of aprepitant analysis, it is recommended to exclude filtration of neutral aqueous solutions, replacing it with centrifugation, or to use acidic dissolution media.