Abstract Background Tenofovir alafenamide (TAF) is a key component of many fixed-dose combinations used to treat HIV. There are limited data on the pharmacokinetics of plasma tenofovir alafenamide (TAF), tenofovir (TFV), or intracellular tenofovir diphosphate (TFV-DP), among people with HIV (PWH) and tuberculosis (TB) who are taking rifampicin-based TB treatment. Methods Participants in the intervention arm of the INSIGHT trial (NCT04734652) receiving bictegravir/emtricitabine/TAF (BIC/FTC/TAF 50/200/25mg) were enrolled into the semi-intensive pharmacokinetic sub-study. BIC/FTC/TAF was administered twice-daily during rifampicin-based TB treatment (∼24 weeks) and once-daily thereafter. Plasma (TAF/TFV) and dried blood spot samples (TFV-DP) were collected at weeks 4 and 12 (pre-dose, 1, 2, 4, 6, and 8-12h post-dose) during TB treatment and at week 32 (pre-dose, 1, 2, 4, 6-8 and 24-25h post-dose) post-TB treatment. Pharmacokinetic parameters were determined using non-compartmental analysis. Clinical and safety data were collected. Results Among 43 participants enrolled; median (IQR) age and weight were 35 (30-39) years and 58 (52-65) kg; 77% were male. Geometric least square mean ratios (90% CI) at week 12 (twice-daily TAF) relative to week 32 (once-daily TAF) for TAF AUC0-4, TFV and TFV-DP AUC0-24 were 1.55 (1.13-2.13), 1.24 (1.07-1.44), and 1.32 (1.13-1.53), respectively. At week 24, 95% of participants achieved viral suppression, with no treatment-related serious adverse events or drug discontinuations. Conclusion Twice-daily TAF was safe and efficacious and achieved similar exposures of intracellular TFV-DP in PWH taking rifampicin for TB compared to once-daily TAF taken alone. These data support the use of TAF in a fixed-dose combination of BIC/FTC/TAF during rifampicin-containing TB treatment.