An approach for optimization of epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitors using truncated thienopyrimidine structures combined with enzymatic assay has been evaluated. This was done by synthesis and EGFR activity measurement of a series of fragment structures and their corresponding drug-model analogues. On average, the activity of the drug-like structures increased a ten-fold as compared to the fragments. However, the potency of the drug-model compound could not be precisely predicted, visualising the typical challenge with linking substructures. Additionally, the activity data provided useful SAR information with respect to stereochemical preference and the structure requirements of the 4-amino group. A retrospective evaluation of binding efficiency in previously discovered thieno[2,3-d]pyrimidines, suggests a high probability of obtaining potent EGFR inhibitors if based on the 3-chloro-4-fluoroaniline moiety. Compounds derived from (S)-2-hydroxy-1-phenylethylamine also have resulted in highly active EGFR-TK inhibitors. In contrast the 3-ethynylaniline containing structures appears more difficult to develop. Thieno[2,3-d]pyrimidin-4-amines have also been used for construction of irreversible EGFR-TK inhibitors. The data indicate these compounds to possess sub-optimal non-covalent interactions.