A non-invasive method for measurement of the bladder wall nonlinear elastic behavior is presented. The method is based on acoustoelasticity modeling of the elasticity changes in bladder tissue modulus at different volumetric strain levels. At each volume, tissue strain is obtained from the real-time ultrasound images. Using acoustic radiation force, a transient Lamb wave is excited on the bladder wall and instantaneous modulus of shear elasticity is obtained from the 2-D Fourier analysis of the spatial-temporal dispersion maps. Measured elasticity and strain data are then used in an acoustoelasticity formulation to obtain the third order elastic coefficient, referred to as nonlinearity parameter A, and initial resting elasticity μ0. The method was tested in ex vivo porcine bladder samples (N = 9) before and after treatment with formalin. The estimated nonlinearity parameter, A, was significantly higher in the treated samples compared to intact (p < 0.00062). The proposed method was also applied on 16 patients with neurogenic bladders (10 compliant and 6 non-compliant subjects). The estimated nonlinearity parameter A was significantly higher in the non-compliant cases compared to the compliant (p < 0.0293). These preliminary results promise a new method for non-invasive evaluation of the bladder tissue nonlinearity which may serve as a new diagnostic and prognostic biomarker for management of the patients with neurogenic bladders.