Axoneme structures in sperm tail, is a supramolecular protein complex with motor protein and regulatory which playing a crucial role in determining sperm motility. Dynein, one of the three members of cytoskeletal motor protein, has a ring of six AAA+ which linked together into one large polypeptide that contribute to the formation of sperm flagella bending. The previously research reported that the first two AAA motor domains, AAA1 and AAA2, were a major site in ATP hydrolysis associated with motility in the flagellum. Intrauterine Insemination (IUI) as a management of infertility requires sperm preparation procedures, by Swim-up (SU) and Density Gradient Centrifugation (DGC), in order to enhance the quality regarding concentration and motility of the initial sperm. This study aimed to evaluate the efficiency of the DGC and SU methods in selecting sperm, based the expression of sperm dynein AAA1 and AAA2. Semen samples were obtained from men underwent sperm preparation for IUI and divided into two groups, normozoospermia and asthenozoospermia, according to World Health Organization 2010 guideline. Semen analysis was performed to measure the sperm motility and velocity, before and after sperm preparation. The axoneme was isolated from the obtained samples from SU and DGC methods, while the level of AAA1 and AAA2 was measured by ELISA. This study showed that the percentage of motile sperm and velocity of prepared sperm in both groups in prepared sperm (post-SU and post-DGC) was higher compared to whole semen. The expression of sperm dynein AAA1 of prepared sperm in normozoospermia group showed higher, while in asthenozoospermia group showed lower activities compared to whole semen. The expression of sperm dynein AAA2 of prepared sperm in both groups showed lower activities compared to whole semen. The sperm preparation enhanced the quality of sperm and may increase the expression of sperm dynein AAA1 compared to the whole semen, without the involvement of sperm dynein AAA2.