ObjectivesAcute hemorrhage can cause significant morbidity and mortality arising from trauma, bleeding disorders, surgical procedures, or obstetric complications. Surgical hemostasis methods may fail to stop acute bleeding due to the complex bleeding dynamics of each bleeding type. Therefore, developing safe and effective topical hemostatic agents remains crucial. The human amniotic membrane (hAM) has established clinical evidence of effectiveness in promoting wound healing and tissue regeneration. Despite its unique biological and immunologic properties and its structural composition of established hemostatic elements, the hemostatic role of hAM has not been yet explored. The present study aimed to investigate this potential role and to describe the development protocol and characterization of hAM‐derived topical hemostat.MethodsSurface electron microscope (SEM) imaging and Fourier transform infrared (FTIR) spectroscopy were used for characterization, and mouse models with induced peritoneal and tail wound bleeding were employed to evaluate the hemostatic effectiveness using physiological studies, in comparison to a chitosan‐based combat‐scale hemostat.ResultsThe hAM hemostat showed a distinctive composition by SEM and FTIR. Applying equal masses of the hAM hemostat, the commercial hemostat, or a combination reduced peritoneal wound bleeding time to averages of 108.4, 86.2, and 76.8 s, respectively, compared to the control group (300 s). Tail wound bleeding times were similarly reduced with no significant difference between the hAM and the commercial hemostat (P values = 0.29, 0.34 in peritoneal and tail wounds, respectively). Neither hemostat affected coagulation time.ConclusionThis study describes a simple cost‐effective preparation protocol for a hAM‐based hemostatic agent. The long‐recognized safety, sustainability, and immunotolerance advantages of hAM can establish superiority over commercial hemostats with reported safety concerns. Robust research validation in larger‐scale bleeding models is required for wider applications and severe bleeding types.