OBJECTIVE Validation of a test method is critical for confirming that the test can generate accurate and precise data. Although commercial biochemical test kits exist there are no specific and validated commercial clinical chemistry test kits designed for horses. The aim of this study was to validate commercial clinical chemistry test kits designed for a human serum for use in horses. RESULTS Blood samples were collected from 29 apparently healthy adult male horses and pooled serum was prepared. Validation comprises replication and recovery experiments. Total observable error (TEo), sigma (σ) metrics, and quality goal index (QGI) were used to support the validation studies. Intra- and inter-assay variability was 2.05% and 2.08%, 2.26% and 1.89%, 2.4% and 1.63%, for total cholesterol, urea and total protein, respectively; recovery was 99.46%, 97.32%, and 100.1% for total cholesterol, urea and total protein, respectively. TEo% for the specified analytes was within the total allowable error (TEa). All three analytes satisfied the recommended requirement (> 3σ). The QGI for urea, as it had below 6σ was 0.95 indicating imprecision and inaccuracy. The results endorse the suitability of the studied commercial test kits and illustrated the acceptance criteria for horse's serum.