Due to the diversities in design and structure of bikes, different cycling competitions were driven. Among those different types of cycling, road bike cycling is the most popular. This article will introduce serial physiological test methods for road bike cyclists. Most test methods mentioned in the current article are to physiological tests designed by Australia National Sports Training Center for cyclists, including measurements of body composition, maximum oxygen uptake, heart rate test and lactic acid threshold test and etc. Technically, a step-by-step protocol as well as professional comment were included for each test method in purpose of providing domestic cyclists and coaches a reference to objectively evaluate their training efficiency as well as competitive performance.