Salvadora persica plant having a number of antimicrobial substances and the roots of the S. persica shrub have been demonstrated to possess antimicrobial activity. Sticks from the roots of S. persica have been used for centuries as a traditional method of cleaning teeth. Benzyl isothiocyanate is the main antimicrobial agent found in the root of the plant. Here, we developed high-performance thin-layer chromatography (HPTLC) and reversed-phase high-performance liquid chromatography (RP-HPLC) methods for the qualitative analysis of benzyl isothiocyanate in the S. persica root powder and the dental care herbal formulation labeled to contain Miswak extract. A HPTLC method was carried out using pre-coated silica gel aluminum TLC plates 60 F254, and the mobile phase consisting of n-hexane (10 mL) furnished compact spots at RF 0.39 ± 0.01. A HPLC method was carried out using a column Phenomenex C18(150 mm × 4.6 mm, 5 μm) and methanol‒10 mm ammonium acetate (70:30, v/v) as the mobile phase with a good retention time, 8.179 ± 0.05. The developed method was validated by HPTLC in accordance with the International Council of Harmonization (ICH) Q2 (R1) guidelines for precision, accuracy, and robustness. The developed plate was scanned and quantified densitometrically at 250 nm. Linear regression analysis revealed a good linear relationship between the peak area and the amount of benzyl isothiocyanate in the range of 3–15 μg per band (r2 = 0.997). Fingerprinting was done by HPTLC and HPLC method by using a dental care herbal formulation labeled to contain Miswak extract. The proposed method will be useful to evaluate the therapeutic efficacy of Miswak extract and dental care herbal formulations containing Miswak extract.