Clinicians commonly recommend increased hydration to patients with voice disorders. However, effects on clinical voice outcome measures have been inconsistent. Hydration-induced change within different layers of vocal fold tissue is currently unknown. Magnetic Resonance Imaging (MRI) is a promising method of noninvasively measuring water content in vocal folds. We sought to image and quantify changes in water content within vocal fold mucosa and thyroarytenoid muscle after dehydration and rehydration. Excised porcine larynges were imaged using proton density (PD) weighted MRI (1) at baseline and (2) after immersion in one of five hypertonic, isotonic, or hypotonic solutions or in dry air. Larynges dehydrated in hypertonic solutions or dry air were rehydrated and imaged a third time. Scans revealed fluid-rich vocal fold mucosa that was distinct from muscle at baseline. Baseline normalized signal intensity in mucosa and muscle varied by left vs. right vocal fold (p < 0.01) and by anterior, middle, or posterior location (p < 0.0001). Intensity changes in the middle third of vocal fold mucosa differed by solution after immersion (p < 0.01). Hypertonic solutions dehydrated the middle third of mucosa by over 30% (p < 0.001). No difference from baseline was found in anterior or posterior mucosa or in muscle after immersion. No association was found between intensity change in mucosa and muscle after immersion. After rehydration, intensity did not differ by solution in any tissue, and was not different from baseline, but post-rehydration intensity was correlated with post-immersion intensity in both mucosa and muscle (p < 0.05), suggesting that degree of change in vocal fold water content induced by hypertonic solutions ex vivo persists after rehydration. These results indicate that PD-MRI can be used to visualize large mammalian vocal fold tissue layers and to quantify changes in water content within vocal fold mucosa and thyroarytenoid muscle independently.