Air Traffic Control (ATC) is a complex cognitive task. The complexity may cause the workload of air traffic controllers (ATCs) to increase. Increased workload could degrade operator performance, which further affect safety and functioning of the system. Numerous studies in evaluating mental workload in various tasks used physiological and biochemical measures, including studies in ATCs. The use of physiological measures in those studies provides unique information of the operator’s condition. This systematic review summarizes literatures on the measurement of the mental workload in ATCs using physiological and biochemical measures. The conducted systematic search come up with thirty-four studies to include for analysis. Physiological measures are categorized into cardiovascular, ocular, brain, respiration, and skin measures. Biochemical measures consisted of cortisol and salivary immunoglobulin (sIgA). The literature review covers various studies either in simulation environment or real working environment, all with different conditions and task scenarios. Even though this review specifically focuses on mental workload of ATCs, in general, the result of physiological measures still differs between broad studies. The difference might be influenced by the study task loads, difficulty levels, and the participants’ characteristics.