Acute respiratory tract infections (ARTIs) are the most common diseases worldwide and an important cause of morbidity and mortality. Upper and lower respiratory tract infections are caused by a variety of bacterial, viral, and fungal pathogens. Clinical diagnosis of respiratory tract infections is challenging because of indistinguishable symptoms. Laboratory diagnosis is performed by serology, culture, electron microscopy, and immunological antigen detection assays. These conventional diagnostic procedures are time consuming, lack sensitivity, require special laboratory setup, and well-trained staff. In the clinical laboratories, multiplex molecular nucleic acid amplification tests (NAATs) are continuously replacing conventional diagnostic methods. This review summarizes and discusses the availability, clinical use, advantages and disadvantages of multiplex molecular methods in the detection and identification of ARTI pathogens. The multiplex molecular assays can simultaneously detect 20 or more bacterial and viral pathogens and have the advantage of increased sensitivity, specificity, and rapid turn-around time. These assays are helpful in syndromic based testing in high risk patient population, particularly those who are immunocompromised, hospitalized and/or seen in the emergency department. Limitations of multiplex molecular assays include inability to detect all the possible pathogens that can be present, not being able to differentiate between asymptomatic carriers and true infections, and sometimes results are difficult to interpret. Furthermore, additional testing may be required as these assays do not provide any information regarding antimicrobial susceptibility profile. Rapid and accurate diagnosis of respiratory tract infections leads to better treatment decisions, reduction in the further diagnostic procedures, length of hospital stay, better infection control measures, and associated healthcare costs.