This study evaluated the effect of using different bio-relevant formulations as the dissolution medium in the DissolvIt® system. The fluids investigated were polyethylene oxide with lipids (PEO), Survanta® and an in-house developed simulated lung lining fluid (sLLF). A Flixotide 50 µg Evohaler® was used to generate an aerosol, which was deposited on cover slips using PreciseInhale®. The fluticasone propionate (FP) particles dissolved into the bio-relevant medium and drug was transferred into a flow-though perfusate within the DissolvIt system. FP as quantified by LC-MS/MS following solid phase extraction (SPE) of the samples. Excellent linearity (R2 = 0.999) was observed. The inter-day and intra-day precision data complied with the validation guidance for bioanalytical methodologies. Differences in FP concentration in the perfusate when using PEO and sLLF were observed at 20 min; 0.29 ± 0.09 and 0.12 ± 0.02 %/mL respectively (p≤0.05). The FP concentration in the perfusate was similar when using PEO and Survanta. It was concluded that the LC-MS/MS assay was validated successfully and provides a sensitive method to quantify FP in dissolution studies. The hypothesis that dissolution of FP in the perfusate would be enhanced in sLLF was not supported by this study. These findings may be attributable to FP residing preferentially in lipid structures in sLLF, limiting FP availability for transfer into the perfusate. Further studies are required to assess whether using a more bio-relevant media provides a more predictive measure of inhaled particle dissolution.