Antibodies are critical tools in medicine and research, and their affinity for their target antigens is a key determinant of their efficacy. Traditional antibody affinity maturation and interaction analyses are often hampered by time-consuming steps such as cloning, expression, purification, and interaction assays. To address this, we have developed FASTIA (Fast Affinity Screening Technology for Interaction Analysis), a novel platform that integrates rapid gene fragment preparation, cell-free protein synthesis, and bio-layer interferometry with non-regenerative analysis. Using this approach, we can analyze the intermolecular interactions of over 20 variants over 2 days, requiring only the parent protein expression plasmid and basic equipment. We have demonstrated the ability of FASTIA to discriminate between single-domain antibody variants with different binding affinities using the anti-HEL VHH antibody D2-L29, and mapped the results to the crystal structure to identify key interaction sites. FASTIA provides results comparable to those obtained using traditional methods. Our system bypasses the need for genetic engineering facilities and can be easily adopted by laboratories, accelerating the protein engineering and optimization processes. In addition, FASTIA is applicable to other protein-protein interactions, making it a versatile tool for studying molecular recognition. FASTIA facilitates efficient affinity maturation, protein engineering, and analysis of protein-protein interactions. This provides a rapid and accessible route for improving antibodies and a broader understanding of protein interactions.