Plant growth relates to gravity, ensuring that roots grow downwards into the soil and shoots expand aerially. The phytohormone auxin mediates tropistic growth responses, such as root gravitropism. Gravity perception in the very tip of the roots triggers carrier-dependent, asymmetric redistribution of auxin, leading to differential auxin responses and growth regulation at the upper and lower root flanks. This cellular, asymmetry-breaking event will eventually lead to root bending towards the gravity vector. Here, we show how to investigate auxin signaling and auxin carrier dynamics during root gravitropic response, using a chambered cover glass in combination with a confocal live cell imaging approach. To exemplify this method, we used established lines expressing transcriptional and translational green fluorescent protein (GFP) fusions to the auxin responsive promoter element DR5rev and the prominent auxin carrier PIN-FORMED2 (PIN2), respectively. Transgenic seedlings were placed and grown in the chambered cover glasses, enabling defined gravitropic stimulations prior to imaging. This method is optimal for inverted microscopes and significantly reduces stressful manipulations during specimen preparation.