When the rotary blood pumps are used as ventricular assist devices, the pump flow rate will have a pulsatile component even at a constant impeller rotational speed due to the remaining beating of the natural heart. However, previous studies on the in vitro hemolysis evaluation of a rotary blood pump have always been conducted under steady states and this pulsation was not taken into account. In this study, the hemolysis in a centrifugal blood pump is evaluated under the pulsatile flow condition in vitro. The required time-varying flow rate is obtained by conducting a system simulation of the pump-assisted cardiovascular system, and realized by controlling a pulsation unit in the experiments. The results of our tests indicate a significant increase in hemolysis under the pulsatile flow condition compared with the non-pulsatile condition. To reveal the flow characteristics responsible for the higher hemolysis, transient computational fluid dynamic simulations are then performed. This study suggests that traditional hemolysis evaluation under the steady states may not fully represent the hemolytic performance in the clinical use. For the ventricular assist pumps at the design stage, eliminating the concern about the extra hemolysis under the pulsatile condition will be helpful for the subsequent in vivo experiments.