Assessment of the unbound pharmacologically active fraction (fu; as the ratio of unbound to total concentration) of dolutegravir could improve therapeutic drug monitoring (TDM) in patients that experience virological failure or toxicity, despite receiving adequate total concentrations. This study evaluated (i) dolutegravir's fu through equilibrium dialysis (ED), (ii) the pre-analytical parameters that influence fu, and (iii) fu's inter-individual variability in HIV patients. Validation of the LC-MS/MS method followed FDA guidelines. The results, based on coefficients of variation (results from nominal concentrations <15%), allowed accurate measurement of unbound and total dolutegravir concentrations. Equilibrium during ED was obtained in 4h. Sparse non-specific binding (9%) was observed, allowing results interpretation without interference. Steps before analysis (e.g., conservation at +4°C, freeze/thaw cycles) did not influence fu, allowing easy integration of fu analysis within laboratory routines. Anticoagulants from samples (citrated versus heparinized; p<0.001) and hemolysis (p=0.007) influenced fu and could lead to misinterpretation. Developed was then performed to the HIV-patients' plasma (n=54). Results, expressed as median InterQuartileRange[25%;75%] were 0.45% IQR[0.38;0.55] for fu, 9.26μg/L IQR[4.62;15.14] for unbound, and 2035μg/L IQR[878.5;2640] for total concentration. The high inter-individual variability observed in the unbound form from HIV patients was a first step towards integrating dolutegravir TDM.