Subharmonic aided pressure estimation (SHAPE) is a technique that utilizes subharmonic signals from microbubble contrast agents for pressure estimation. Validation of the SHAPE technique relies on synchronous measurements of in vivo pressures using contrast microbubbles and a pressure catheter (reference standard). For the guidance and placement of pressure catheter in vivo, iodinated contrast is used with fluoroscopy. Therefore, during data acquisition for validation studies of the SHAPE technique, both contrast microbubbles and iodinated contrast are present simultaneously within the vasculature. This study aims to elucidate the effects of iodinated contrast (Visipaque, GE HealthCare) on subharmonic signal amplitude from contrast microbubbles (Definity, Lantheus Medical Imaging, Inc.). In an acrylic water tank, 0.06 mL of Definity and varied amounts of Visipaque (0.14, 0.43, 0.85, and 1.70 mL) were added to 425 mL of deionized water. Ultrasound scanning was performed with a SonixTablet scanner (BK Medical Systems) using optimized parameters for SHAPE with Definity ( ftransmit/receive = 3.0/1.5 MHz; chirp down pulse). Subharmonic data was acquired and analyzed at 9 different incident acoustic outputs ( n = 3). Results showed an increase in subharmonic signal amplitude from Definity microbubbles in the presence of 0.14 mL Visipaque by 2.8 ± 1.3 dB ( p < .001), no change with 0.85 mL Visipaque (0.7 ± 1.2 dB; p = .09) and a decrease in subharmonic amplitude in the presence of 1.70 mL Visipaque by 1.9 ± 0.7 dB ( p < .001). While statistically significant effect on subharmonic signal amplitude of Definity microbubbles was noted due to the mixture, the magnitude of the effect was minimal (~2.8 dB) and unlikely to impact in vivo SHAPE measurements.