Cryopreservation of milt sample refers to preserving these samples by storing them at excessively low temperature (−196 °C) in order to increase their shelf-life. One of the main objectives of this project was to standardize and optimize the dilution methods and activation of milt samples before analysis of motility by CASA. The optimization was performed on fresh milt from Atlantic salmon and cryopreserved milt from Atlantic salmon and Atlantic halibut. Further, the aim was to elucidate possible correlation between the objective sperm quality parameters and fertilization rate. The sperm quality attributes were assessed following the protocols of evaluation of sperm motility by CASA, viability by flow cytometry and ATP content by bioluminescence assay. From the optimized methods, Dilution F with activator C was chosen for motility analysis of fresh salmon milt samples, Dilution S with activator C for cryopreserved salmon milt samples and Dilution S with activator S for cryopreserved halibut milt samples in the further work. The obtained results were processed statistically by linear mixed model and two-way ANOVA in order to know the effect of activator and dilution method on sperm motility parameters along with their corresponding interaction on these parameters. The optimal time frame upon thawing of the cryopreserved milt samples until motility analysis was also done with the selected activator and dilution methods. For cryopreserved salmon milt, the optimal time frame was concluded to be within six minutes and within four minutes for cryopreserved halibut milt sample. The viability and ATP of salmon milt from eight individuals filled into Squarepacks were found to be higher with one of the individuals, 66.53% and 1118.51 nM respectively. However, this was not reflected in the higher fertilization rates. Further, correlation results of salmon Squarepacks showed that only sperm velocity parameters with low sperm to egg ratio were significantly associated with the fertilization rate. Based on the results obtained, it can be concluded that Dilution S can be favoured over Dilution U for cryopreserved milt samples. The activator C and S can be used for activation of cryopreserved salmon and halibut milt, respectively. Moreover, low sperm to egg ratios revealed stronger statistical results for association between in vitro milt quality parameters and fertilization outcome. However, in order to demonstrate the possible relationship between fertilization rate and the sperm quality parameters, further work and investigation is required.