Monitoring dynamic load transfer from train traffic to sub-ballast layers is crucial for verifying the reliability and safety of railway lines, assessing the design cost-effectiveness and achieving minimum environmental impact. For this purpose, measurements in labs, in situ or modeling the influence of dynamic loads on the immediate and long-term roadway quality are often performed using suitable software. The available test sections enabled monitoring of the dynamic loads and optimizing the critical spots where increased dynamic effects from railway traffic may occur. The subject of this paper is the calibration of the sensors installed in the different test sections of the trans-European corridor number V. As a result, the necessary input parameters for the subsequent numerical modeling of the dynamic effects on the track substructure and vibration propagation on the available sections of the upgraded railway line were obtained. The sensor calibration was carried out on the experimental field, part of the Experimental Basis of the Department of Railway Engineering and Track Management. As part of the calibration, the sensitivity of the sensors embedded in the track bed to the applied dynamic loads resulting from the impact effects of the lightweight deflectometer was assessed. The result of the calibration was the demonstration of sufficient sensitivity of the sensors and their suitability for implementation in an actual railway track structure, with the aim of obtaining relevant values of the response of the sub-ballast layers to dynamic loads and assessing the operational impacts on the sustainable environment. Also, the main result of the research was the possibility of using the theoretical–experimental route to optimize the layers of the railway body.