Efavirenz requires adequate serum concentrations (1–4 µg/mL) to ensure therapeutic efficacy and avoid central nervous system toxicity. Due to significant interindividual pharmacokinetic variability, therapeutic drug monitoring (TDM) is often necessary. Reliable bioanalytical methods for quantifying efavirenz in biological matrices are therefore essential. Previous dried blood spot (DBS) methods using High-Performance Liquid Chromatography–Photodiode Array (HPLC–PDA) showed suboptimal performance and lacked compliance with current bioanalytical guidelines. This study aimed to develop and validate an improved HPLC–PDA method for efavirenz quantification in DBS using an internal standard. The optimized sample preparation employed protein precipitation and methanol extraction, followed by chromatographic separation under isocratic conditions with UV detection at 245 nm. The method demonstrated excellent linearity (r2 = 0.9995) within 0.1–30 µg/mL, accuracy within ±9.3%, and precision (CV < 4.6%). Recovery ranged from 91.4–95.7%, and no significant carryover or matrix effect was observed. Validation results met the acceptance criteria specified by the US FDA (2018) and EMA (2022) guidelines. The proposed method offers enhanced sensitivity, accuracy, and precision, supporting its suitability for routine TDM of efavirenz to improve treatment safety and effectiveness. © 2026 Putri et al.