We have studied noninvasive devices for measuring total hemoglobin and hemoglobin derivatives such as carboxyhemoglobin (COHb) and methemoglobin (MetHb). A calibration procedure needs to be developed to evaluate or calibrate these devices and pulse oximeters for clinical practice. However, people and animals are sometimes exposed to risk when they are used for calibration. In this paper, we propose a new in vitro calibration system for a pulse photometer. This system has a novel double-layer pulsation flow-cell that incorporates both venous and arterial blood flow. Using the calibration system, we are able to measure the in vitro pulsatile optical density ratio (Φvt). The measured Φvt agrees well with the in vivo pulsatile optical density ratio (Φvi). This system simulates an in vivo environment with high accuracy and enables safe calibration. Consequently, the calibration system is able to standardize the performance and accuracy of pulse photometry.