One of the mechanisms of healthy hepatocytes to detoxify ammonia in the liver consists of converting it into urea. When liver function is impaired, this detoxification capacity decreases and may cause severe pathologies, such as hepatic encephalopathy. Consequently, urea synthesis is a parameter that can be used to monitor liver functionality. In this chapter, a protocol for the measurement of urea synthesis in the culture medium of cultured rat hepatocytes is described. The procedure relies on a chromogenic reagent that specifically forms a colored complex with urea. The latter can be measured colorimetrically and is directly proportional to the urea concentration in the sample.