Purpose: To characterise a biorelevant simulated lung fluid (SLF) based on the composition of human respiratory tract lining fluid. SLF was compared to other media which have been utilized as lung fluid simulants in terms of fluid structure, biocompatibility and performance in inhalation biopharmaceutical assays. Methods: The structure of SLF was investigated using cryo-transmission electron microscopy, photon correlation spectroscopy and Langmuir isotherms. Biocompatibility with A549 alveolar epithelial cells was determined by MTT assay, morphometric observations and transcriptomic analysis. Biopharmaceutical applicability was evaluated by measuring the solubility and dissolution of beclomethasone dipropionate (BDP) and fluticasone propionate (FP), in SLF. Results: SLF exhibited a colloidal structure, possessing vesicles similar in nature to those found in lung fluid extracts. No adverse effect on A549 cells was apparent after exposure to the SLF for 24 h, although some metabolic changes were identified consistent with the change of culture medium to a more lung-like composition. The solubility and dissolution of BDP and FP in SLF were enhanced compared to Gamble’s solution. Conclusion: The SLF reported herein constitutes a biorelevant synthetic simulant which is suitable to study biopharmaceutical properties of inhalation medicines such as those being proposed for an inhaled biopharmaceutics classification system.