This research was motivated by the importance of developing nutritious alternative foods, specifically by utilizing beetroot flour as a source of iron and vitamin C to enhance the nutritional value of processed products. Beetroot flour is a highly nutritious ingredient with the potential to substitute wheat flour in food production. The study aimed to determine the effect of varying beetroot flour substitution levels on iron and vitamin C content, as well as product acceptability based on color, aroma, taste, and texture. A true experimental method was employed using a Completely Randomized Design (CRD) with four formulation treatments: F0 (control), F1, F2, and F3, featuring beetroot-to-wheat flour ratios of 0%, 30%, 50%, and 70% beetroot flour, respectively. Organoleptic testing was conducted with 30 semi-trained panelists. Data were analyzed using ANOVA followed by the Duncan Multiple Range Test (DMRT), while iron (Fe) and vitamin C contents were determined via laboratory analysis. Formulation F0 achieved the highest preference rating, followed by F2 (50% beetroot flour and 50% wheat flour). Laboratory analysis results showed iron (Fe) contents of 0.157 mg/L and 0.193 mg/L (F0) and 0.143 mg/L and 0.153 mg/L (F2), while vitamin C contents were 27.27 mg/g and 28.42 mg/g (F0) and 29.48 mg/g and 29.91 mg/g (F2). The results indicate that the addition of beetroot flour significantly affected the organoleptic characteristics and nutritional content of the cookies (p<0.05). Further research is recommended to optimize the formulation to produce a product that is both highly preferred and nutritionally superior.