This case highlights the effect of heterophilic antibody interference in two siblings with hypopituitarism. Prolactin measurements performed on the UniCel DxI 800 in patients followed up with a diagnosis of hypopituitarism were found to be falsely elevated. For further investigation, polyethylene glycol precipitation (PEG), heterophilic antibody blocking tubes (HBT), serial dilution, and three different blocking antibodies were used. The PEG test showed a reduction of less than 40%, and macroprolactinemia was excluded. In the serial dilution test, the results were not linear, raising suspicion of interference. Lower prolactin levels were reported in three different systems. After HBT application, prolactin levels unexpectedly increased in both siblings. The increase in prolactin levels observed after HBT and the addition of different blocking antibodies could be due to an unexpected interaction between the blocking antibodies and the analysis reagents. The investigations using blocking antibodies were unable to determine the exact nature of the interfering agent.