In drug formulations containing polymer excipients, the effects of the polymer on the dissolved free drug concentration and resulting dissolution or release can be important, especially for poorly soluble drugs. In this study, an in vitro method based on pulsatile microdialysis (PMD) was developed to quantitatively determine dissolved free concentrations of drugs in the presence of polymers in aqueous media in situ (e.g., in place within the system being characterized). Formulations were made by dissolving various ratios of the drug griseofulvin and polymer PVP K30 in water and allowing the mix to equilibrate. A PMD probe was immersed in each mixture and the dissolved free drug concentrations were determined in the PMD samples. The experimental procedure and the equations used for data analysis are presented. To assess the consistency of data, a binding model was fit to the data obtained using PMD by calculating the dissolved free drug fraction fD for each drug–polymer ratio in solution, and obtaining the product of the binding stoichiometry and binding constant (νK per mole of polymer) from the slope of a plot of (1−fD)/fD vs. the molar polymer concentration. For comparison, equilibrium binding experiments were also performed at 23 C, and the determined value of νK was similar to the value found using PMD. Experiments were performed at three temperatures, and a plot of ln (νK) vs. 1/T was linear and a binding enthalpy of −110.9 ± 4.4 J/mol of monomer was calculated from its slope. It was concluded that PMD can be used to determine the dissolved free drug concentrations in situ, which allows characterization of the drug–polymer interaction, even for low drug concentrations. This information may be important in modeling the dissolution or release of drugs from formulations containing polymers.