Exposure to excessive whole-body vibration is linked to health issues and may result in increased rates of mortality and morbidity in infants. Newborn infants requiring specialized treatment at neonatal intensive care units often require transportation by road ambulance to specialized care centers, exposing the infants to potentially harmful vibration and noise. A standardized Neonatal Patient Transport System (NPTS) has been deployed in Ontario, Canada, that provides life saving equipment to patients and safe operation for the clinical care staff. However, there is evidence that suggests patients may experience a higher amplitude of vibration at certain frequencies when compared with the vehicle vibration. In a multi-year collaborative project, we seek to create a standardized test procedure to evaluate the levels of vibration and the effectiveness of mitigation strategies. Previous studies have looked at laboratory vibration testing of a transport system or transport incubator and were limited to single degree of freedom excitation, neglecting the combined effects of rotational motion. This study considers laboratory testing of a full vehicle and patient transport system on an MTS Model 320 Tire-Coupled Road Simulator. The simulation of road profiles and discrete events on a tire-coupled road simulator allows for the evaluation of the vibration levels of the transport system and the exploration of mitigation strategies in a controlled setting. The tire-coupled simulator can excite six degrees-of-freedom motion of the transport system for vibration evaluation in three orthogonal directions including the contributions of the three rotational degrees of freedom. The vibration data measured on the transport system during the tire-coupled testing are compared to corresponding road test data to assess the accuracy of the vibration environment replication. Three runs of the same drive file were conducted during the laboratory testing, allowing the identification of anomalies and evaluation of the repeatability. The tire-coupled full vehicle testing revealed a high level of accuracy in re-creating the road sections and synthesized random profiles. The simulation of high amplitude discrete events, such as speed hump traverses, were highly repeatable, yet yielded less accurate results with respect to the peak amplitudes at the patient. The resulting accelerations collected at the input to the manikin (sensor located under the mattress) matched well between the real-world and road simulator. The sensors used during testing included series 3741B uni-axial and series 356A01 tri-axial accelerometers by PCB Piezotronics. These results indicate a tire-coupled road simulator can be used to accurately evaluate vibration levels and assess the benefits of future mitigation strategies in a controlled setting with a high level of repeatability.