The present study focuses on the High-Performance Thin-Layer Chromatography (HPTLC) analysis of Anastatica hierochuntica L., a medicinal plant known for its traditional use in treating fertility problems, menstrual ailments, etc. The aim was to develop a precise, reproducible, and robust HPTLC method for the qualitative and quantitative analysis of the main bioactive compound Silybin present in the flower. Methanolic extracts of AH were prepared and subjected to HPTLC analysis on silica gel plates using Methanol: Ethyl acetate: Toluene: Formic acid (2.0: 3.0: 5.0: 0.2 v/v/ v/v) as mobile phase. The plates were scanned and densitometric analysis was performed at 291nm to identify and quantify the major phytoconstituent. Silybin was used as a chemical marker in the TLC fingerprinting of the methanol extracts, which resulted in the discovery of spots under UV light. The RF of 0.526 in these spots confirmed the presence of silybin in the AH extract at concentration of 1.076 mg in 10.000 mg. The developed HPTLC method provides a reliable and efficient tool for the standardization and quality control of AH extracts, ensuring their efficacy and safety in herbal formulations.