Background/Objectives: Diet significantly influences gut microbiota (GM), with variations in GM responses linked to the type and quantity of food consumed. These variations underscore the need for personalized nutrition. The Stance4Health (S4H) project developed the S4H Food Frequency Questionnaire (S4H-FFQ) and the i-Diet S4H app to assess dietary intake of foods affecting GM. This study aimed to validate the S4H-FFQ against the validated I.Family-FFQ and the i-Diet S4H app; Methods: The S4H-FFQ, with 200 food items across 14 food groups, evaluates dietary intake over the past month. Qualitative validation compared food group consumption frequencies from the S4H-FFQ and the I.Family-FFQ, while quantitative validation assessed nutrient and energy intake using the i-Diet S4H app. The S4H-GM score, a measure of GM-relevant food consumption, was evaluated through the S4H-FFQ and i-Diet S4H app; Results: Pearson correlations between the S4H-FFQ and the I.Family-FFQ ranged from 0.3 to 0.7 and were statistically significant across all the food groups. Quantitative validation showed lower but consistent correlations, comparable with other studies, confirming the S4H-FFQ’s ability to estimate food intake. A positive correlation was also found between the S4H-GM scores from the S4H-FFQ and the i-Diet S4H app (p < 0.001); Conclusions: The S4H-FFQ is a reliable tool for assessing dietary patterns that influence GM. Its application in nutritional studies can enhance personalized nutrition and support future research aimed at optimizing GM and improving health outcomes.