Objectives: The objective of the paper was to provide an overview of the Regulation on medicines for pediatric use in the European Union and its impact on pediatric clinical trials and the availability of medicines for children, as well as to describe the regulatory framework of clinical trials in the pediatric population. Materials and Methods: During the preparation of the paper, the website of European Medicines Agency (EMA), databases and scientific publications (Pub Med) were searched, and the Regulation on medicines for pediatric use in the European Union was reviewed in detail. Results: This paper provides a comprehensive overview of the regulatory framework of medicines for pediatric use and clinical trials in the pediatric population. Regulation (EC) no. 1901/2006 on medicinal products for pediatric use in the European Union has been described in details, as well as the specifics of clinical trials in children, paying attention to the protection of children's rights as a particularly vulnerable group of potential participants in clinical trials. Conclusion: Over the past two decades, significant efforts have been made in improving the availability and safety of medicines for children within the European Union. A key role represented the Regulation (EC) no. 1901/2006 on medicines for pediatric use from 2007, which established a framework of obligations and incentives for pharmaceutical companies to encourage the development of medicines for children and to improve the availability of information on the use of medicines in the pediatric population. The implementation of the Regulation resulted in an increase in pediatric trials and newly approved drugs for pediatric use. Although there is a notable progress in the development of pediatric medicines after the introduction of the Regulation, there are still challenges and areas for improvement to advance the development of pediatric medicines and meet the therapeutic needs of children.