Orthosiphon stamineus is widely used as an ingredient in traditional medicine and functional food partially for its main active compound, sinensetin. Plant growth and sinensetin contents are sensitive to many variables, including phytogeographical profiles. This study sought to evaluate the quality of O. stamineus obtained from nine locations in Indonesia, predicated on sinensetin levels assessed using TLC-densitometry. Thin Layer Chromatography (TLC) was conducted with silica gel 60 F254 as the stationary phase and toluene: ethyl acetate (5:7) and a drop of formic acid for every 10 ml of that solvent mixture as the mobile phase and was analyzed without a derivatization reagent. The created method proved uncomplicated and satisfied the specificity parameters, as indicated by the identical UV spectrum shared between the sinensetin standard and sample (λmax = 334 nm). Also, it showed good linearity for sinensetin in the range of 14.5–87 ng/band (r = 0.9886). Limits of detection and limits of quantification were 9.03116 and 27.36717 ng/band, respectively. In addition, the method possessed good intra- and interday precision (marked by Relative Standard Deviations (RSDs) of 1.65%–6.47% and 4.97%) and accuracy (95.86, 120.18, and 82.44% recoveries in standard addition with three-level solutions). Of the 14 samples, sinensetin was undetected in two but found in various concentrations in the other 12 samples, from 0.0238 to 0.1533 mg/g. Using a sample from the Tawangmangu area as a reference, three groups of samples were formed: those with lower sinensetin contents (Jakarta Selatan, Lamongan, Jombang, and Sampang), higher sinensetin contents (Surabaya, Mojokerto, Kediri, and Kotabaru), and similar sinensetin contents as the reference sample (Batu, Gresik, and Madiun). The TLC-densitometry designed in this study is straightforward but satisfies the validation parameters; thus, it can be used to qualitatively and quantitatively analyze sinensetin in O. stamineus. Overall, O. stamineus in different phytogeographical zones ...