Purpose. Sentinel lymph node (SLN) biopsy in the surgical treatment of early stage breast cancer is currently employed as part of the routine protocol. Recent studies have shown the use hemosiderin, a new marker derived from the patient’s blood, capable of facilitating the surgical procedure. However, the hypothesis that the blood-derived marker would be hemosiderin arose only from clinical data and preliminary research. This study aimed to study the blood derivative characteristics, leading to clarification of the main substance responsible for SLN marking. Methods. Preclinical tests were conducted in Wistar rats to evaluate SLN tissue marking. After obtaining hemoglobin, the fraction was assessed, and lymphatic mapping and SLN identification of the popliteal region were performed. Drainage time, standard concentration, tissue marking after proteolysis, and thermostability were assayed. Histopathological evaluation by optical microscopy following the animal procedure. Box plots were generated for graphic presentation. Results. It was found that hemoglobin is the main responsible for the labeling of SLN. This labeling process can be optimized by applying 3% nitrite and protease inhibitors to the hemoglobin concentrate for 48h, respecting the drainage time of 2h maintaining the sample temperature at 25°C. Conclusion. This study demonstrates the possible clinical applicability of the hemoderivative protein as a tissue marker in identifying the sentinel lymph node in early and locally advanced breast cancer. The marking process is optimized by standardizing the homeostatic conditions of the biological marker.