Abstract Objectives Dalbavancin is a lipoglycopeptide with a long half-life, making it a promising treatment for infections requiring prolonged therapy, such as complicated Staphylococcus aureus bacteraemia. Free drug concentration is a critical consideration with prolonged treatment, since free concentration–time profiles may best correlate with therapeutic effect. In support of future clinical trials, we aimed to develop a reliable and reproducible assay for measuring free dalbavancin concentrations. Methods The ultracentrifugation technique was used to determine free dalbavancin concentrations in plasma at two concentrations (50 and 200 mg/L) in duplicate. Centrifuge tubes and pipette tips were treated for 24 h before use with Tween 80 to assess adsorption. Dalbavancin concentrations were analysed from the plasma samples (total) and middle layer samples (free) by LC/MS/MS with isotopically labelled internal standard. Warfarin served as a positive control with known high protein binding. Results Measurement of free dalbavancin was sensitive to adsorption onto plastic. Treatment of tubes and pipette tips with ≥2% Tween 80 effectively prevented drug loss during protein binding experiments. By the ultracentrifugation method, dalbavancin’s protein binding was estimated to be approximately 99%. Conclusions Dalbavancin has very high protein binding. Given dalbavancin’s high protein binding, accurate measurement of free dalbavancin concentrations should be a key consideration in future exposure–response studies, especially clinical trials. Future investigations should confirm if the active fraction is best predicted by the free or total fraction.