Two-photon polymerization (TPP) is an advanced 3D fabrication technique capable of creating features with submicron precision. A primary challenge in TPP lies in the facile and accurate characterization of fabrication quality, particularly for structures possessing complex internal features. In this study, we introduce an automated brightfield layerwise evaluation technique that enables a simple-to-implement approach for in situ monitoring and quality assessment of TPP-fabricated structures. Our approach relies on sequentially acquired brightfield images during the TPP writing process and using background subtraction and image processing to extract layered spatial features. We experimentally validate our method by printing a fibrous tissue scaffold and successfully achieve an overall system-adjusted fidelity of 87.5% in situ. Our method is readily adaptable in most TPP systems and can potentially facilitate high-quality TPP manufacturing of sophisticated microstructures. Read more here