A study was carried out at the Biotechnology-cum-Tissue Culture Centre, OUAT, Bhubaneswar, India, to standardise a viable protocol for the “High frequency in vitro cloning of Banana ( Musa acuminata) cv. Grande Naine” during the year 2014. This has helped to determine the best media compositions for shoot multiplication and rooting of the variety Grande Naine, so as to get optimum results with a minimized cost of production. The result revealed that MS medium supplemented with 5.0 mg/l BAP and 1.0 mg/l IAA was found to be a most potent combination and produced a significantly higher number of shoots/explants (4.47) after 30 days of culturing. The same combination recorded significantly higher number of shoots/explants, i.e., 5.97 and 6.13 in 1st and 2nd sub-culturing respectively.MS medium fortified with 2.0 mg/l BAP and 1.0 mg/l IBA was found to be ideal for the early shoot elongation attending height of 3.52 cm with more number of leaves/plants (4.2) and produced more number (5.4) of longer roots (4.47 cm) roots after 30 days of inoculation. Approximately 1308 number of Tissue culture plantlets/explant can be produced by following this protocol.