In many applications, high intensity focused ultrasound (HIFU) treatments are guided using ultrasound imaging. To date, efficacy of ablative technologies such as HIFU for the treatment of liver tumor has been studied in animal models without any tumor or in small animals like rat and rabbit with established tumor. The purpose of this study was to use an in vivo tumour-mimic model for guiding HIFU ablation in the liver. Tumor-mimics were created in in vivo porcine livers by injecting a warm mixture of agarose, cellulose, glycerol and methylene blue, which formed 1 cm hyperechoic discrete lesions on sonograms. In vivo experiments were then conducted in 10 pigs for 30 days. 3.9 plusmn 0.3 (min.3.max.5) tumor-mimics were created in each animal using an open approach to evaluate if they are well tolerated. Lesions were characterized sonographically, examined macroscopically and using histopathologic analysis with reproducible dimensions (major diameter: 13.9 plusmn 1.8 mm (min.8.1.max.22.0 mm)) and similar to those measured on the sample removed during surgery. The average error between macroscopic measurements and ultrasound measurements is 18.8plusmn 7.5 % (min.0.0.max.77.5%) for 1 cc injections. Target creation took a few minutes per tumour-mimic and 80% of injections were successful. Ablations in normal liver tissue and in tumor-mimic were performed in vitro on 20 samples using a HIFU probe (working frequency: 3 MHz) with an integrated ultrasound imaging probe (working frequency: 7.5 MHz). Ablation sizes were not significantly different for controls and tumor-mimics. The agarose-based tissue-mimic creates realistic sonographic targets in vivo and in vitro for ultrasound-guided HIFU ablation in an in vivo model