A rapid and feasible method of HPTLC is standardized for quantification of anethole in essential oil’s extract and from herbal formulations of fennel seed. The developed densitometric HPTLC method was performed to estimate the existence of anethole in the essential oil, extract and herbal formulations of fennel with the optimized concentration of hexane: Ethyl acetate (8:2%, v/v, mobile phase) on glass coated silica gel 60 F254 plates (20 × 10 cm) scanned with the absorbance of λ260 nm under densitometric condition. The Linearity of regressions revealed a satisfactory relationship between peak area and concentration of anethole in between the range of 100-600 ng/spot. This reliable method was validated as per the ICH guidelines to fulfill the necessary parameters such as accuracy and robustness. The amount of anethole in essential oil (0.098 ± 0.002%), extract (0.101 ± 0.004%) and three herbal formulations A (0.024 ± 0.004%), C (0.019 ± 0.002%) while anethole is not detected in B formulations from fennel seed was completely estimated by the developed method. The standardized methods and its validation gave new insights of HPTLC based detection and quantification of anethole in other aromatic plants as well as in other pharmacological formulations.