Although haemolysis is the most common source of preanalytical error in clinical laboratories, its influence on cattle biochemistry remains poorly understood. The effect of haemolysis and its clinical relevance were investigated in 70 samples in which haemolysis was artificially induced (by spiking with increasing amounts of haemolysate, yielding 0.0%, 0.2%, 0.5%, 1.0%, 2.5%, 5.0% and 10% haemolysis degree (HD)), focusing on key parameters for bovine metabolic health assessment, including albumin, alkaline phosphatase (ALP), aspartate aminotransferase (AST), blood urea nitrogen (BUN), calcium (Ca), cholesterol, creatinine, creatine kinase (CK), gamma-glutamyl transferase (GGT), globulins, magnesium (Mg), phosphorus (P), total bilirubin (TBIL) and total proteins (TP). Preanalytical haemolysis significantly affected most (8 of 14) of the biochemical parameters analysed, leading to significant increases in concentrations of albumin (starting at 5% HD), cholesterol (at 5% HD) and P (at 10% HD) and to significant decreases in Ca (at 2.5% HD), creatinine (at 5% HD), globulins (at 10% HD), TBIL (at 2.5% HD) and TP (at 10% HD). Comparison of the present and previous data indicated that, for each parameter, the HD required to produce significant bias and the clinical relevance of over- and underestimation are variable and appear to depend on the analytical technique used. Therefore, different laboratories should evaluate the influence of haemolysis in their analytical results and provide advice to clinicians accordingly. Affected parameters should be interpreted together with clinical signs and other analytical data to minimize misinterpretations (false or masked variations). Finally, due to the significant impact on numerous parameters and the limited potential for correction, we recommend rejection of samples with >10% HD.