Firmness is a very important quality property for kiwifruit. This property is affected by storage temperature and duration. Recently, some destructive and non-destructive methods were developed and used for fruit firmness assessment. However, non-destructive methods have merits of real-time and online applications as well as preventing fruit destruction. The objective of the present study was to evaluation of non-destructive acoustic technique for assessing the kiwifruit firmness at two different temperature levels during a storage period. On this basis, firmness of kiwifruit during two weeks of storage by non-destructive method was monitored. For determining the correlation between resonant frequency, stiffness and elasticity coefficients, and fruit firmness, destructive puncture test (Magness- Taylor) was also applied. The maximum penetration force in puncture test depicted a strong correlation with those from acoustic tests, resonant frequency, stiffness coefficient and elasticity modulus with the coefficients of determination of 0.838, 0.857 and 0.865, receptivity. According to the maximum correlation coefficient between modulus of elasticity and firmness, the relation was verified by new set of data, and revealed that it could estimate the fruits firmness with an error of 7.4 percent. Therefore, nondestructive acoustic test is an appropriate substitution method for estimation of kiwifruit firmness during shelf-life.