Background: Isoniazid (INH) remains a first-line drug for the treatment of tuberculosis (TB) in young children. In 2010, the WHO recommended an increase in the daily dose of INH up to 10 (7–15) mg/kg. Currently, there are no INH suspensions available in Europe. Methods: We aimed to characterize the pharmacokinetics of a licensed INH suspension (10 mg/mL, Pharmascience Inc., Montreal, QC, Canada) in children receiving INH daily at 10 mg/kg in a single-center, open-label, non-randomized, phase IIa clinical trial (EudraCT Number: 2016-002000-31) in Barcelona (Spain). Samples were analyzed using a validated UPLC-UV assay. The N-acetyltransferase 2 gene was examined to determine the acetylation status. A non-compartmental pharmacokinetic analysis was conducted. Results: Twenty-four patients (12 females) were included (primary chemoprophylaxis, n = 12; TB treatment, n = 9; and TB infection preventive treatment, n = 3). The acetylator statuses were homozygous fast (n = 3), heterozygous intermediate (n = 18), and homozygous slow (n = 2; unavailable in one patient). The INH median (IQR) C_max and AUC_0–24h values were 6.1 (4.5–8.2) mg/L and 23.0 (11.2–35.4) h∙mg/L; adult targets (>3 mg/L and 11.6–26.3 h∙mg/L) were not achieved in three and six cases, respectively. Gender, age at assessment (2 years), and INH monotherapy (vs. combined TB treatment) had no impact on pharmacokinetic parameters. Significant differences in C_max (p = 0.030) and AUC_0–24h (p = 0.011) values were observed based on acetylator status. Treatment was well tolerated, and no severe adverse events were observed; three patients developed asymptomatic mildly elevated alanine aminotransferase levels. Conclusions: In infants and children receiving a daily INH suspension at 10 mg/kg, no safety concerns were raised, and the target adult levels were reached in the majority of patients.