Abstract: Overexpression of the human epidermal growth factor receptor 2 (HER2) is a well-established diagnostic and prognostic biomarker in certain malignancies and is generally associated with aggressive metastatic progression and poor clinical outcome. HER2-directed diagnostic and therapeutic strategies have achieved substantial clinical success, with monoclonal antibodies remaining the current gold standard. Nevertheless, this approach is limited by several important factors, including complex and expensive manufacturing processes and the relatively low stability of antibodies. These limitations underscore the need for alternative targeting agents. Among the most promising alternatives are affibodies, a class of small engineered proteins derived from the Z domain of staphylococcal protein A. In particular, the ZHER2 affibody family constitutes a promising group of small proteins that exhibit high affinity for the target receptor and have demonstrated success in realworld clinical applications of diagnostics and therapy of HER2overexpressing tumors. In this report, we summarize the development and optimization history of these affibodies, their structural features, advantages and limitations for clinical applications. In addition, we present our own original data highlighting the utility of ZHER2 affibodies for in vitro diagnostics of malignant tumors.