The present study was carried out to investigate the impact of different substrates on the primary and secondary hardening of tissue culture raised banana plantlets. Among eight different treatment combinations of potting mixtures, treatment PHT7 which contained cocopeat with the combination of perlite and vermiculite (2:1:1) emerged as the best potting media with the survival percentage of 98.90% with comparative better growth during primary hardening. Simultaneously maximum growth and development including plant height (15.36 cm), number of leaves (5.71), pseudostem girth (3.88 cm), maximum number of roots (5.90), maximum root length (3.69 cm) has been observed during primary hardening. For secondary hardening, eight combinations of substrates were used in conjunction with soil. Among all the treatments in the secondary hardening, SHT4 (soil+ bonemeal; 3:1) along with the application of Jeevamrit have shown highest field survivability (100%). Moreover a maximum shoot length (33.40 cm) has been observed during secondary hardening with maximum number of leaves were recorded (9.25) with pseudo stem girth 6.98 cm. The outcome of the present study suggested that the treatment PHT7 performed better growth during primary hardening. However the treatment SHT4 has shown comparatively higher growth during secondary hardening.