To the authors’ knowledge, there is currently no literature or guidance recommendation regarding whether the dose of dolutegravir (DTG) should be increased when co-administered with darunavir/ritonavir (DRV/r) in patients with acute human immunodeficiency virus infection (AHI). This study assessed the pharmacokinetics (PK) of twice-daily (BID) DTG and once-daily (QD) DRV/r, and compared this with DTG QD without DRV/r in patients with AHI. Forty-six participants initiated antiretroviral therapy within <24 h of enrolment: DTG 50 mg BID, DRV/r 800/100 mg QD, and two nucleoside reverse transcriptase inhibitors (NRTIs) for 4 weeks (Phase I); and DTG 50 mg QD with two NRTIs thereafter (Phase II: reference). Total DTG trough concentration (C trough ) and area under the concentration–time profile of 0–24 h (AUC 0–24h ) were predicted using a population PK model. DTG glucuronidation metabolic ratio (MR) and DTG free fraction were determined and compared per treatment phase using geometric mean ratio (GMR) and 90% confidence interval (CI). Participants had a predicted geometric mean steady-state DTG C trough of 2.83 [coefficient of variation (CV%) 30.3%] mg/L (Phase I) and 1.28 (CV% 52.4%) mg/L (Phase II), with GMR of 2.20 (90% CI 1.90–2.55). Total exposure during DTG BID increased but did not double [AUC 0–24h GMR 1.65 (90% CI 1.50–1.81) h.mg/L]. DTG glucuronidation MR increased by approximately 29% during Phase I. DTG C trough was above in-vivo EC 90 (0.32 mg/L) during both phases, except in one participant during Phase I. At Week 8, 84% of participants had viral loads ≤40 copies/mL. The drug–drug interaction between DTG (BID) and DRV/r (QD) was due to induced glucuronidation, and is not clinically relevant in patients with AHI.