Shampoo is a cosmetic preparation that can be used to clean hair, so hair and scalp become clean. Water spinach can be used to treat dandruff. This study aims to determine the anti-dandruff activity of shampoo preparations from water spinach ethanol extract. This study used 4 shampoo formulas, with variations in the concentration of water spinach ethanol extract by 8% (formula 1), 9% (formula 2), 10% (formula 3). And 0% (negative control). Test the physical properties of the preparation include organoleptic tests (odor, shape, and color), pH measurement, viscosity test, foam height test, and anti-dandruff activity test. Data analysis was statistically tested using ANOVA. The results showed the higher the concentration of the extract, the better the results will be seen from the parameters of the viscosity test and foam height test. While the effect of extract concentration on each of the physical properties of the four anti-dandruff shampoo formulas showed good stability in terms of organoleptic parameters and pH. The results of the anti-dandruff activity test showed that the anti-dandruff shampoo with 10% water spinach extract anti-dandruff shampoo had the best antifungal activity compared to formula 1 with extract concentration of 8% and formula 2 with extract concentration of 9%.