Objective In-situ sentinel lymph node (SLN) detection is  an important component in staging cancers of various origins. At present, technetium-99m(^99mTc)-labeled nanoparticle formulations like sulfur colloid and human serum albumin (HSA) nanocolloid are used in the clinic as SLN tracers. In India, HSA nanocolloid cold kits have so far been imported. This study aims to develop and evaluate an indigenous alternative to imported HSA nanocolloid cold kits for SLN detection/imaging. Materials and methods Production of cold kits was standardized and the product was characterized for its suitability in terms of particle size. ^99mTc-labeling of an in-house HSA nanocolloid was optimized, and the yield and stability of the product were assessed. Animal studies were performed in Wistar rats using the footpad model. Clinical evaluation was performed in 54 patients using a combination of scintigraphic imaging and a hand-held gamma probe. Results and conclusion With the optimized protocol, HSA nanocolloids with particle sizes ranging from 50 to 200 nm were obtained. Greater than 90% ^99mTc-labeling yield was obtained in 15 min reactions, and the radiopharmaceutical was stable for up to 24 h after preparation. The animal studies showed similar SLN uptake and improved retention pattern compared with those of the imported Nanocoll radiopharmaceutical. Clinical studies showed detectable ‘hot’ nodes in 53 of 54 patients, demonstrating sensitivity of the product for clinical utility. In conclusion, this indigenous HSA nanocolloid cold kit is proposed as a logistically favorable alternative to imported kits for SLN detection in the Indian clinical scenario.