This study was designed to assess the effect of different concentrations of L-carnitine and Co enzyme Q10 (COQ10) supplied to SMART Pro-medium on human sperm motility and sperm grade activity (progressive, non-progressive and immotile) during in vitro sperm activation. Eighty seven samples of semen of infertile and fertile men were randomly collected from the subjects at the High Institute for Infertility Diagnosis and Assisted Reproductive Technologies / Al-Nahrain University. The mean age of the men was (32.034 ± 0.57) years with mean duration of infertility (4.644 ± 0.25) years. Seminal fluid analyses were done involving macroscopic and microscopic examinations according to WHO criteria. Swim-up technique was dependent for in vitro sperm activation , the washed samples were divided before using centrifugation swim-up technique into 3 groups: control group (G1) without Lcarnitine and CoQ10. While, in G2, G3 two concentrations of L-carnitine and CoQ10 (20 µg , and 40 µg) were used. Moreover, all groups within post-activation enriched with SMART-Pro media. The sperm motility and sperm grade activity were evaluated after the addition of L-carnitine and COQ10. The results showed a significant (P<0.05) differences were showed in sperm motility and sperm grade activity of post-activation when compared with the preactivation. In G2 and G3, with dose of (20 µg and 40 µg) of L-carnitine and CoQ10, the results showed a significant (P<0.05) increase in the percentages of sperm motility, progressive sperm motility in spite of the increase in the percentages of sperm motility, progressive sperm motility in G3 compared to G2, there was non significant (P>0.05) differences between G2 and G3. The results for non-progressive sperm motility% and total progressive sperm motility millions/ mL. for 40 µg (G3) groups were significantly (P< 0.05) decreased when compared to control group (G1) with non-significant (P>0.05) differences between 20µg (G2) group, with significant (P< 0.05) decreased in immotile sperm percentage in post-activation when compared to the pre-activation. From the results of the study, it can be concluded that the addition of 40 µg of L-carnitine and CoQ10 to washed sperms can improve sperm motility and sperm grade activity in vitro. Data were analyzed statistically using complete randomized design (CRD) (one way ANOVA).