The atomic force microscope (AFM) colloid probe technique was utilized to measure cohesion forces (separation energy) between three drug systems as a function of relative humidity (RH). The subsequent data was correlated with in vitro aerosolization data collected over the same RH range. Three drug-only systems were chosen for study; salbutaniol sulphate (SS), triamcinolone acetonide (TAA), and di-sodium cromoglycate (DSCG). Analysis of the AFM and in vitro data suggested good correlations, with the separation energy being related inversely to the aerosolization performance (measured as fine particle fraction, FPFLD). In addition, the relationship between, cohesion, RH, and aerosolization performance was drug specific. For example, an increase in RH between 15% and 75% resulted in increased cohesion and decreased FPFLD for SS and DSCG. In comparison, for TAA, a decrease in cohesion and increased FPFLD was observed when RH was increased (15-75%). Linear regression analysis comparing AFM with in vitro data indicated R-2 values > 0.80, for all data sets, suggesting the AFM could be used to indicate in vitro aerosolization performance. (c) 2006 Wiley-Liss, Inc.