The ileocolonic-targeted delivery of vitamins can establish beneficial alterations in gut microbial composition. Here, we describe the development of capsules containing riboflavin, nicotinic acid, and ascorbic acid covered with a pH-sensitive coating (ColoVit) to establish site-specific release in the ileocolon. Ingredient properties (particle size distribution, morphology) relevant for formulation and product quality were determined. Capsule content and the in vitro release behaviour were determined using a HPLC-method. Uncoated and coated validation batches were produced. Release characteristics were evaluated using a gastro-intestinal simulation system. All capsules met the required specifications. The contents of the ingredients were in the 90.0–120.0% range, and uniformity requirements were met. In the dissolution test a lag-time in drug release of 277–283 min was found, which meets requirements for ileocolonic release. The release itself is immediate as shown by dissolution of the vitamins of more than 75% in 1 h. The production process of the ColoVit formulation was validated and reproducible, it was shown that the vitamin blend was stable during the production process and in the finished coated product. The ColoVit is intended as an innovative treatment approach for beneficial microbiome modulation and optimization of gut health.