Protein-based biogenic materials provide important inspiration for the development of high-performance polymers. The fibrous mussel byssus, for instance, exhibits exceptional wet adhesion, abrasion resistance, toughness and self-healing capacity-properties that arise from an intricate hierarchical organization formed in minutes from a fluid secretion of over 10 different protein precursors. However, a poor understanding of this dynamic biofabrication process has hindered effective translation of byssus design principles into synthetic materials. Here, we explore mussel byssus assembly in Mytilus edulis using a synergistic combination of histological staining and confocal Raman microspectroscopy, enabling in situ tracking of specific proteins during induced thread formation from soluble precursors to solid fibres. Our findings reveal critical insights into this complex biological manufacturing process, showing that protein precursors spontaneously self-assemble into complex architectures, while maturation proceeds in subsequent regulated steps. Beyond their biological importance, these findings may guide development of advanced materials with biomedical and industrial relevance.