Background Dosing recommendations for treating childhood tuberculosis (TB) were revised by the World Health Organization, yet so far, pharmacokinetic studies that have evaluated these changes are relatively limited. We evaluated plasma drug concentrations of rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA), and ethambutol (EMB) among children undergoing TB treatment in Tanzania when these dosing recommendations were being implemented. Methods At the end of intensive-phase TB therapy, blood was obtained 2 hours after witnessed medication administration to estimate the peak drug concentration (C2h), measured using high-performance liquid chromatography or liquid chromatography–tandem mass spectrometry methods. Differences in median drug concentrations were compared on the basis of the weight-based dosing strategy using the Mann–Whitney U test. Risk factors for low drug concentrations were analyzed using multivariate regression analysis. Results We enrolled 51 human immunodeficiency virus–negative children (median age, 5.3 years [range, 0.75–14 years]). The median C2hs were below the target range for each TB drug studied. Compared with children who received the “old” dosages, those who received the “revised” WHO dosages had a higher median C2h for RIF (P = .049) and PZA (P = .015) but not for INH (P = .624) or EMB (P = .143); however, these revised dosages did not result in the target range for RIF, INH, and EMB being achieved. A low starting dose was associated with a low C2h for RIF (P = .005) and PZA (P = .005). Malnutrition was associated with a low C2h for RIF (P = .001) and INH (P = .001). Conclusions Among this cohort of human immunodeficiency virus–negative Tanzanian children, use of the revised dosing strategy for treating childhood TB did not result in the target drug concentration for RIF, INH, or EMB being reached.