In this chapter, an isocratic reverse phase HPLC determination of mimosine was developed and validated in an anti-psoriatic topically applied formulation “Lajjalu”. The chromatography was performed on a C18 column with water-orthophosphoric acid (98.8:0.2, υ/υ) as a mobile phase with a pH of 3.0 at a flow rate of 1.0 mL min−1. Detection was performed at 284 nm, and a sharp peak was obtained for mimosine at a retention time of 2.62 ± 0.01 min. Linear regression analysis data for the calibration plot showed a good linear relationship between response curve and concentration in the range of 0.050–5000 ng mL−1 and the regression coefficient was 0.9998 with the linear regression equation y = 4766.8x−17726. The detection (LOD) and quantification (LOQ) limits were 10.3 and 35.6 ng mL−1, respectively. The wide linearity range, sensitivity, accuracy, short retention time, and simple mobile phase imply the method is suitable for routine quantification of mimosine with high precision and accuracy.