A concentration of 11% of glycerol is the standard one for sperm cryopreservation in chickens, however, the presence of just 2% glycerol already causes severe fertility reduction, suggesting the necessity of removing glycerol before artificial insemination (AI). The major approach developed for this purpose is serial dilution followed by centrifugation (SDC), which demands special equipment (such as a refrigerate room) to maintain post-thaw semen at 4.C, besides being time consuming. Therefore, we attempted to develop a simple method to remove glycerol from chicken frozen-thawed semen based on a colloidal gel, Percoll, which is ordinarily used to select motile and viable sperm in mammals as well as in fresh chicken semen. In this study, we used a Percoll based glycerol removal solution (GRS) containing sucrose to avoid frozen-thawed sperm suffering from osmotic stress. Subsequently, several conditions including GRS compositions (GRS A, B, C and D) and centrifugation temperatures (4 and 20 degrees C) were compared by their influence on sperm in vitro parameters. Afterwards, GRS A and D were selected for fertility evaluation, compared to conventional SDC method. Our results showed that the fertility with GRS A at both 4 and 20 degrees C were higher than GRS D (p < 0.05) and similar or even superior to the fertility obtained with SDC method. Altogether, our novel GRS protocol is a valuable method for chicken sperm cryobanking policy, supported by its notable results of fertility as well as saving 44% of time, with a simple equipment at flexible operation temperatures of 4 or 20 degrees C.