Electroporation is a highly efficient delivery method for transformation in various cell types; however, in microalgae there is lack of optimized electroporation parameters due to cell wall, protoplast preparation and viability. Therefore, we optimized electroporation conditions for transforming microalgae using Chlamydomonas reinhardtii strains of wild type and mutant (cell wall deficient). To investigate the effects of molecule size, calcein (623 Da) and fluorescein isothiocyanate-dextran (FITC-dextran, 40 kDa) were used and various electroporation parameters were applied such as different voltage and pulse length and molecule uptake pattern and cell viability were observed. Cell wall is insignificant in case of small sized molecule uptake as noticed by 1.25 kV/cm and 30 ms for both strains, whereas for larger molecules by 1.5 and 2 kV/cm and 30 ms for mutant and wild type, respectively. In terms of viability, there was no significant difference in both the strains on applied electroporation parameters. The controlled parameters corresponding to 1.5 to 2.0 kV/cm and 20 to 30 ms could be used to deliver macromolecules (DNA, proteins) into cells effectively.