Silent gastric ulceration occurs without evidence of clinical signs and is common in horses. There is currently no a simple and effective method to diagnose this disease. Proteomics can be used to identify serum biomarkers, but the most abundant serum protein, albumin, could conceal candidate biomarkers. Therefore, it is recommended to remove albumin before a proteomic study; however, there is no specific albumin depletion kit or standard protocol available for horse samples. The objectives of this study were to optimise a protocol to remove equine serum albumin and to use albumin-depleted serum to identify the protein biomarkers for silent gastric ulceration. Gastroscopy was used to identify gastric ulceration, and serum was obtained from horses with either a healthy gastric mucosa or gastric ulceration. Serum albumin was removed using the trichloroacetic acid (TCA) protein precipitation method, and this protocol was optimised by varying the concentration of TCA, type of organic solvents, ratio of serum to protein precipitation solution, and incubation times. Electrophoresis and image analysis were used to compare the amounts of albumin, immunoglobulins G (IgG), and protein degradation before and after TCA precipitation. The best protocol was chosen to remove albumin for a proteomic study (electrophoresis and mass spectrometry). The results revealed that protocol 2 (ratio of serum to solution 1:5, 10% TCA in acetone, and 90 min incubation) was the most efficient protocol to remove albumin (98%) and IgG heavy (80%) and light (98%) chains without degrading other proteins. After electrophoresis and mass spectrometry analysis, KRT1, KRT6A and KRT18 were identified as potential markers for silent gastric ulceration.