Assessing dietary habits with validated questionnaires is crucial for achieving reliable results in health research. The aim of this study was the development and validation of a semi-quantitative food frequency questionnaire (FFQ) in an 18–30-year-old Serbian population. A total of 93 participants completed the FFQ and three 24 h dietary recalls (24 hR). Pearson and intraclass correlations between FFQ and 24 hR intakes were assessed and were de-attenuated and energy-adjusted. Bland–Altman plots were generated, and quintiles of energy, water, fiber, and macronutrient intake were analyzed with contingency tables. Adjustments for sex differences were included. The validity of the FFQ varied, with significant correlations for energy, carbohydrates, cholesterol, and vitamin B12, vitamin C and vitamin D. Misclassification rates were generally low. Bland–Altman plots indicated good agreement between methods. It can be concluded that the validated FFQ is a promising tool for dietary assessment in the Serbian population. Results for several nutrients align closely with previous studies. The new FFQ is a useful tool for dietary assessment in the Serbian population.