At present, there are few in vivo experimental studies on anterior chamber flow field, and the relevant technologies are not mature. This study explores the experimental method and key techniques of particle image velocimetry (PIV) for the in vivo measurement of anterior chamber flow field with slow flow velocity in the rabbit with acute intraocular hypertension. The experimental process can be divided into three parts: model construction of rabbit eye with acute intraocular hypertension, in vivo eyeball preparation, and PIV setup. The following key techniques were mainly investigated: the optimal injection strategy of fluorescent particles and the correction strategy for image acquisition errors caused by the effects of image refraction and respiration. The results showed that the best injection method was that 15 μL of fluorescent particles solution was slowly injected into the anterior chamber through the lower part of iris and then the rabbit was released and waited for 13 h. In this way particles were completely distributed in the anterior chamber with the help of the aqueous humor circulation, and then in vivo PIV experiment could be performed. The eyeball should be covered with a square flume filled with ultrasonic coupling gel for the sake of imaging during the experiment. The Maximal Information Coefficient algorithm could be applied to correct the measured results before post-processing calculation. The results indicated that feasible injection strategy of fluorescent particles and the correction strategy for image acquisition are critical to obtain nice experiment effects for the in vivo PIV measurement of anterior chamber flow field in the rabbit with acute intraocular hypertension.