In March 2024, an infection with highly pathogenic avian influenza (HPAI) virus subtype H5N1 of the HA clade 2.3.4.4b was detected in dairy cows in Texas. Following this first detection, the virus was identified in multiple states in the United States, with spillover into other mammals. This raised concerns in the Netherlands that despite having an effective monitoring and surveillance system, H5N1 virus infections in Dutch cattle could have occurred. The aim of this study was therefore to retrospectively screen Dutch cattle for HPAI virus infections for the period between 2022 and 2024. A total of 2,190 archived cattle serum samples from 367 different herds were selected, with an average of 6 samples per herd. These samples were tested using an influenza A blocking ELISA. A Luminex H5/H7 assay was used to confirm and subtype ELISA-positive samples. The influenza A blocking ELISA detects antibodies against all influenza A virus subtypes, whereas the Luminex assay can differentiate antibodies against the surface proteins haemagglutinin (HA or H) 5 and 7 (H5/H7), and neuraminidases (NA or N) 1 to 9 (N1-N9). Four samples (0.2%) reacted positive in the influenza A blocking ELISA, all from different herds located in different parts of the Netherlands. However, all 4 samples tested negative in the Luminex H5/H7 assay, indicating that the samples did not have antibodies against H5, H7, and N1 to N9. The 99.8% specificity of the ELISA may explain the ELISA-positive results. The ELISA used in this study is a multispecies test that has not yet been validated for use in cattle. The duration of detectable antibodies in cattle is unknown, as is the sensitivity to detect past infections with influenza A virus subtypes in cattle for the Dutch situation. Therefore results should be interpreted with caution. The lack of detected antibody responses in cattle in the Netherlands from 2022 to 2024 aligns with the absence of any indication for clinical cases in Dutch cattle over recent years in the national monitoring and surveillance system.