A series of novel pyrazolo[3,4-d]pyrimidine derivatives have been designed based on chemical modifications on the general features of the reported and clinically used EGFR-TKIs such as repalcing of quinazoline moiety of reported EGFR-TKIs as erlotinib by pyrazolo[3,4-d]pyrimidine nucleus,introducing different hydrophobic moieties including phenyl, aromatic heterocyclic, fused aromatic or aliphatic structures , introducing different linkers which may be one atom , two atoms, three atoms,  four atoms and five atoms  and introducing of phenyl ring at position-3 of 1H-pyrazolo[3,4-d]pyrimidine nucleus to occupy the hydrophobic region II of ATP binding site. All the new synthesized compounds were biologically screened in vitro for their cytotoxic activities against four cancer cell lines namely, HepG-2, MCF-7, HCT-116, and Hela. The results of cytotoxic evaluation indicated that compound VI was found to be the most prominent broad-spectrum cytotoxic activity and significantly more potent than doxorubicin with IC50 values of 6.18, 6.48, 4.03, and 5.82μM against tested cell lines. In addition, compounds IXa,b displayed promising cytotoxic effect against all tested cell lines with IC50 values less than 30 μM compared with doxorubicin as a control drug. Besides, compound X possessed excellent anti-proliferative activities against the four cell lines with IC50 values ranging from 18 μM to 39.5 μM. Structural pharmacophoric features indicated that pyrazolo[3,4-d]pyrimidine scaffold having a four atoms linker as thiosemicarbazide moiety as compounds IXa,b which substituted with aliphatic moiety at the 4-position was more potent than those possessing one atom, two atoms, three atoms  and five atoms linkers which lead to significant decrease in cytotoxic