The toxicokinetics (TK) of hydrolyzed fumonisin B1 (HFB1) were evaluated in 16 broiler chickens after being fed either a control or a fumonisins-contaminated diet (10.8 mg fumonisin B1, 3.3 mg B2 and 1.5 mg B3/kg feed) for two weeks, followed by a single oral (PO) or intravenous (IV) dose of 1.25 mg/kg bodyweight (BW) of HFB1. Fumonisin B1 (FB1), its partially hydrolyzed metabolites pHFB1a and pHFB1b, and fully hydrolyzed metabolite HFB1, were determined in chicken plasma using a validated ultra-performance liquid chromatography–tandem mass spectrometry method. None of the broiler chicken showed clinical symptoms of fumonisins (FBs) or HFB1 toxicity during the trial, nor was an aberration in body weight observed between the animals fed the FBs-contaminated diet and those fed the control diet. HFB1 was shown to follow a two-compartmental pharmacokinetic model with first order elimination in broiler chickens after IV administration. Toxicokinetic parameters of HFB1 demonstrated a total body clearance of 16.39 L/kg·h and an intercompartmental flow of 8.34 L/kg·h. Low levels of FB1 and traces of pHFB1b were found in plasma of chickens fed the FBs-contaminated diet. Due to plasma concentrations being under the limit of quantification (LOQ) after oral administration of HFB1, no toxicokinetic modelling could be performed in broiler chickens after oral administration of HFB1. Moreover, no phase II metabolites, nor N-acyl-metabolites of HFB1 could be detected in this study.