The epidermal growth factor (EGF) receptor is commonly targeted in cancer therapy because it is overexpressed in many malignant cells. However, a general problem is to couple the targeting moieties and the drug molecules in a way that results in a homogeneous product. Here, we overcome this issue by engineering a variant of EGF with a single conjugation site for coupling virtually any payload. The recombinant EGF variant K-EGF was expressed in Rosetta with a 4-6 mg/L yield. To confirm the accessibility of the introduced functional group, the ligand was equipped with a sulfo-cyanine dye with a loading of 0.65 dye per ligand, which enables tracking in vitro. The kinetics and affinity of ligand-receptor interaction were evaluated by enzyme-linked immunosorbent assay and surface plasmon resonance. The affinity of K-EGF was slightly higher when compared to the wild-type EGF ( : 5.9 vs. 7.3 nM). Moreover, the ligand-receptor interaction and uptake in a cellular context were evaluated by flow cytometry and quantitative high-content imaging. Importantly, by attaching heterobifunctional polyethylene glycol linkers, we allowed orthogonal click-conjugation of the ligand to any payload of choice, making K-EGF an ideal candidate for targeted drug delivery.