In this present study, the goal was to characterize previously generated rationally shuffled AAV vector library in terms of vector productivity, in vitro transduction efficacy and in vivo biodistribution. Most of the rationally shuffled capsids were successfully produced (48 out of 57) being in the productivity range of the AAV8 vector. AAV vector preparations have been used for in vitro screening on HuH7 cell line and in vivo screening in C57BL/6 male mice. In vitro screening did not reveal any capsids with the potential to outperform AAV8 acceptor capsid while in vivo screening results showed the most pronounced increase in muscle and CNS tropism followed by a moderate increase in liver tropism. Organs that did not display any increase in tropism were the spleen, thymus and kidney. A moderate to a strong positive correlation between RLU/mg values and VGCN/cell has also been demonstrated. In conclusion, a general increase in tissue tropism that has been observed for rationally shuffled capsids together with independent seroprevalence study in human sera that did not reveal any seroprevalence increase serve as proof of the effectiveness of the rational shuffling approach in generating AAV vectors with improved tropism properties and unaffected seroprevalence. The characterization of this library of capsids not only allowed to validate the rational shuffling method but also paved the way for the development of a new generation of capsids with tailored features for gene therapy application. Noteworthy, some characterized capsids might be valuable candidates for gene therapy aiming to target muscles and CNS and their properties will be further investigated in additional animal models.