Biomechanical characterization of cells is becoming one of the non-invasive techniques to monitor the different physical phenomenon of cells including cell growth, proliferation, migration and adhesion on micropatterned substrates. The present study demonstrates micropillars patterned-Polydimethylsiloxane (PDMS) substrate to investigate the biophysico-mechanical properties of normal, cancer and drug-treated cancer cells. The compositions of PDMS were varied to explore the effect of substrate rigidity providing important signals to change in cell-cell and cell-substrate interactions on micropillar substrates. Further, micropillars of different PDMS compositions were fabricated through lithography followed by softlithography technique. Subsequently, normal HaCaT cells, untreated and doxorubicin-treated MDA-MB 231 & MCF-7 breast cancer cells were cultured on without any protein coated micropillar substrates for 24 h that individually act as a force sensor. Deflection of pillar allows the direct visualization and quantification of cell traction forces applied to the pillar using python image processing algorithm developed by us. Results revealed cell focal adhesion site on micropillar varied among HaCaT, untreated and treated MDA-MB 231 & MCF-7 cancerous cells. It was observed HaCaT cells were adhered at (20-26)% of pillars height, however, both untreated cancer cells adhered above 68% but after drug treatment cancer cells adhesion site shifted at (35-55)% of pillar height from bottom. Therefore, changes in the deflection magnitude and traction force with cell types are attributed to the alterations in their different location of cell adhesion site that constitute different mechanical properties of cells. Thus, traction force exerted by various cell lines due to their different biophysico-mechanical properties exploring new insights and advances in cell mechanics and differentiation of normal and cancer cells as well as drug effect on cancer cells and in cancer therapeutics.