A series of 3,19-O-acetal derivatives of andrographolide (1) have been synthesized by protecting the hydroxyls at C-3 and C-19 through a novel route. All the derivatives were evaluated for in vitro anticancer activity. Among the synthesized derivatives, compounds 3, 3a, 3d, 3e, 7 and 8 showed potential cytotoxicity against human cancer cell lines A549 (lung), HeLa (cervical), ACHN (renal), B-16 (melanoma) and IEC-6 (small intestine). The binding mode conformation was evaluated through docking simulations, while bioavailability/drug-likeness was evaluated through predictive ADME screening studies. All the derivatives were characterized by spectroscopy and the stereochemistry of compounds 3a and 3e was also confirmed by X-ray analysis.