Thin-layer chromatography (TLC) is an often omitted analytical technique due to its lower sensitivity and separation capacity. Even in the era of high-performance liquid chromatography (HPLC), thin-layer chromatography still offers many advantages, such as simplicity, rapidity, and cost-effectiveness, which predict TLC to be the first-choice method for the laborious optimization process requiring analysis of numerous samples. In this work, a thin-layer chromatography method with chemical and densitometric detection was used to optimize a dispersive liquid-liquid microextraction (DLLME) process for the extraction and preconcentration of estradiol in human urine. The chromatographic system consisted of silica gel plates as the stationary phase and toluene-ethanol (9:1; v/v) mixture as the developing solvent. The plates were dyed with 10% phosphomolybdic acid reagent and sequentially evaluated densitometrically at λ = 430 nm. In the context of DLLME optimization, parameters including the type and volume of extraction and dispersive solvents, centrifugation, salt addition and extraction time, were studied. The proposed DLLME-TLC method was successfully applied to the determination of estradiol in real human urine samples.