The quantification of drug in living cells is of increasing interest in pharmaceutical research because of its importance in understanding drug efficacy and toxicity. Label-free in situ measurement methods are advantageous for their ability to obtain chemical and time profiles without the need of labelling or extraction steps. We have previously shown that Fourier transform infrared (FTIR) spectroscopy has the potential to quantify drug in situ within living cells at micromolar level when a simple solution of drug was added to the medium. The purpose of this study was to demonstrate that the approach can evaluate more complex systems such as the effect of membrane modification by a formulation on drug uptakes. The inhaled corticosteroid, beclomethasone dipropionate (BDP), in Calu-3 respiratory epithelial cells in the absence and presence of glycerol, an excipient in some inhaled medicines was used as the model system. The FTIR method was first validated for limit of detection (LOD) and quantification (LOQ) according to published guidelines and the LOQ was found to be ∼ 20 μM, good enough to quantify BDP in the living cell. The uptake of BDP by living Calu-3 cells was found to be reduced in the presence of glycerol as expected due to the stiffening of the cell membrane by the presence of glycerol in the formulation. This study demonstrates the valuable analytical capability of live-cell FTIR to study the effect of formulation on drug transport in lungs and to evaluate drug availability to intracellular targets. We conclude that FTIR has potential to contribute widely at the frontier of live-cell studies.