This study aimed to improve the detection of erythropoietin (EPO) and its biosimilars in human urine using the SAR-PAGE technique by electrophoresis method in pre-casted gels to help differentiate each biosimilar of EPO according to their molecular masses along with their electrophoretic characters. 20 ml human urine samples were collected, purified then spiked with the known concentration of erythropoietin standards. SAR-PAGE electrophoresis was applied using two different concentrations of N-Lauroylsarcosine sodium salt to visualize the difference and effect of sarkosyl on human erythropoietin detection, followed by immunodetection using chemiluminescence reagent. All data were collected, tabled, and statistically analyzed. The first group (using 0.1% w/v SARCOSYL-PAGE) showed large condensed shadowed bands and asymmetric bands behavior while the second group (using 0.12 % w/v SARCOSYL-PAGE) showed confirmed, thin,sharp bands and symmetric identical bands behavior. Increasing sarcosyl concentration to 0.12 % w/v affect the immunological detection by increasing the affinity of the monoclonal anti-EPO antibody to the protein chain in both PEGylated and non-PEGylated epoetins thus leading to enhanced antibody binding and a sharper electrophoretic separation and detectable bands.