Breast cancer is one of the most common malignant tumors found in women and hasbecome as the second cause of death in women after cervical cancer.We successfully synthesizedtechnetium-99m labeled Human Serum Albumin nanoparticle(99mTc-HSA nanoparticle); a radiopharmaceuticalwhich potentially used for application in lymphoscintigraphytechnique for tracing lymphatic system.This study was aims to obtain pre-clinical data from 99mTc-HSA nanocolloidin Sprague Dawleyfemale ratsgiven7, 12-dimethylbenzeneanthracene (DMBA) - induced cell proliferation in the breast tissues.The rats are divided into two groups, one groupsaregiven DMBA and observed for 5, 8 and 12 weeks and the other group is without any treatment as control.Biodistribution study of 99mTc-HSA nanoparticlewas conducted to identify the accumulation of this agent in the popliteal and lumbar lymph nodes and the other tissuecompare to normal rats. 99mTc-HSA nanoparticleshown good accumulation in popliteal lymph node 2.90+1.32 %ID; 0.81 + 0.47 %IDand 1.04+0.46 %ID at one hour post injectionin 5, 8 and 12 weeks respectively.The popliteal extraction (PE) was estimated with value 87.92 %, 74.07%, 71.79% in 5, 8 and 12 weeks respectively. This result showed that99mTc-HSA nanoparticleis very promising compound to be lymphoscintigraphyagentin detection of breast cancer.Key words: lymphoscintigraphy, 99mTc-HSA nanoparticle, breast cancer