Therapeutic affinity proteins offer a targeted mode of action due to their typically high affinity and specificity for disease-associated molecules. In cancer therapy, such target molecules are often overexpressed receptors on tumor cells. However, their presence in healthy tissues can lead to on-target, off-tumor toxicity, necessitating strategies to enhance tumor selectivity. Here, we present an affibody-based prodrug concept that exploits tumor-associated proteases for selective activation. As proof of concept, we designed, produced, and characterized HER2-specific prodrug candidates, each incorporating a distinct protease substrate for selective activation by tumor-associated proteases. Their activation by corresponding proteases and subsequent HER2 binding were assessed. The most promising prodrug candidate was conjugated to the cytotoxic agent DM1 and evaluated for cytotoxicity in HER2-positive cancer cells. The results demonstrated potent, HER2-dependent cell killing, with markedly reduced cytotoxicity in the absence of prodrug activation. These findings support the feasibility of affibody-based prodrugs as a strategy to enhance tumor selectivity and minimize off-tumor toxicity in targeted cancer therapy.