In this thesis four approaches to increase the quality and assure the validity of three-dimensional (3D) models generated within the single particle cryo-electron microscopy (CryoEM) workflow are introduced. ProteoPlex is a high-throughput technique and software tool for the evaluation of the most stabilizing biochemical conditions of macromolecular complexes. The validation technique 3D correlation of tilted pairs (3DCTP) can be applied alongside the single particle refinement cycle to detect model bias utilizing tilted pair images. A new analysis method identifies the presence of overfitting in high resolution CryoEM 3D structures. Finally, a set of criteria covering different model quality aspects of the generated 3D structure is introduced.