Liposome-supported peritoneal dialysis (LSPD) with transmembrane pH-gradient liposomes was previously shown to enhance ammonia removal in cirrhotic rats and holds promise for the treatment of hyperammonemic crises-associated disorders. The main objective of this work was to conduct the preclinical evaluation of LSPD in terms of pharmacokinetics, ammonia uptake, and toxicology to seek regulatory approval for a first-in-human study. The formulation containing citric acid-loaded liposomes was administered intraperitoneally at two different doses once daily for ten days to healthy minipigs. It was also tested in a domestic pig model of hyperammonemia. The pharmacokinetics of citric acid and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was linear following intraperitoneal administration of medium and high dose. There was no systemic accumulation following daily doses over ten days. The systemic exposure to phospholipids remained low. Furthermore, the liposome-containing peritoneal fluid contained significantly higher ammonia levels than the liposome-free control, demonstrating efficient ammonia sequestration in the peritoneal space. This was indeed confirmed by the ability of LSPD to decrease plasmatic ammonia levels in artificially induced hyperammonemic pigs. LSPD was well tolerated, and no complement activation-related pseudoallergy reactions were observed. The safety profile, the linear pharmacokinetics of citric acid following repeated administrations of LSPD as well as the linear dose-dependent ammonia sequestration in the peritoneal space provide a strong basis for the clinical investigation of LSPD.