This study introduces microscale thermophoresis (MST), a sample-efficient, free-solution method for analyzing protein-protein and protein-small-molecule interactions in biological liquids such as blood serum or cell lysate. MST is based on the thermophoresis of molecules, which provides information about molecule size, charge, and hydration shell. The technique was validated using immunologically relevant systems, including human interferon gamma and calmodulin with calcium. The affinity of quercetin, a small-molecule inhibitor, to its kinase PKA was determined in buffer and human serum, revealing a 400-fold reduced affinity in serum. This study demonstrates that MST can provide valuable insights into the influence of the biological matrix on protein functionality and facilitate more efficient drug development. The method is highly sensitive, works in free solution, and minimizes sample consumption and contamination. It also allows for the analysis of interactions in complex biological liquids, providing a more accurate representation of in vivo conditions.This study introduces microscale thermophoresis (MST), a sample-efficient, free-solution method for analyzing protein-protein and protein-small-molecule interactions in biological liquids such as blood serum or cell lysate. MST is based on the thermophoresis of molecules, which provides information about molecule size, charge, and hydration shell. The technique was validated using immunologically relevant systems, including human interferon gamma and calmodulin with calcium. The affinity of quercetin, a small-molecule inhibitor, to its kinase PKA was determined in buffer and human serum, revealing a 400-fold reduced affinity in serum. This study demonstrates that MST can provide valuable insights into the influence of the biological matrix on protein functionality and facilitate more efficient drug development. The method is highly sensitive, works in free solution, and minimizes sample consumption and contamination. It also allows for the analysis of interactions in complex biological liquids, providing a more accurate representation of in vivo conditions.