Enabling direct and definitive free fraction determination for highly-bound compounds in protein binding assay

•A customized protein binding assay approach is developed for free fraction determination of highly protein-bound compounds, featuring 24 -h incubation for equilibrium dialysis, followed by microflow LC–MS/MS for post-assay bioanalysis.•Longer incubation time than typically used, ensures highly protein-bound compounds reaching dialysis equilibrium, while utilization of microflow LC–MS/MS provides far better analytical sensitivity needed by the low-concentration dialysate samples, than conventional LC–MS/MS .•Further analytical sensitivity can be achieved by utilizing the flexible and accurate injection volume on the microflow LC–MS/MS system, while maintaining the linear response in detecting all the samples for valid free fraction determination.•The combined approach enables direct and definitive free fraction determination for highly -bound compounds, hence the interspecies comparison and triaging of the test compounds.