This study explores the possibility of formulation of a cold kit for fast and easy preparation of a PET radiopharmaceutical, ⁶⁸Ga-NOTA-UBI (29-41) for clinical translation. In this study, Circular dichroism (CD) spectroscopy to study conformation of NOTA-UBI (29-41) and its comparison with conformation of UBI (29-41) was done. Pharmaceutical grade cold kits of NOTA-UBI (29-41) were formulated for radiolabeling with 68Ga and necessary quality control tests were carried out. ⁶⁸Ga-NOTA-UBI (29-41) could be prepared in >90% radiochemical yield and radiochemical purity using cold kits of NOTA-UBI (29-41). In vitro and in vivo evaluation of ⁶⁸Ga-NOTA-UBI (29–41) was done to demonstrate specificity of the agent for imaging infection. Kits were utilized for preparation of patient dose of ⁶⁸Ga-NOTA-UBI (29-41). Simple instant thin layer chromatography (ITLC) method for estimating radiolabeling yield of ⁶⁸Ga-NOTA-UBI (29-41) at hospital radiopharmacy was demonstrated. Clinical evaluation was done in patients with suspected infection. 148–185 MBq of ⁶⁸Ga-NOTA-UBI (29–41) was injected intravenously in three patients.  ⁶⁸Ga-NOTA-UBI (29-41) uptake could clearly delineate infection foci from non target normal tissues. This is the first report on formulation of a cold kit of NOTA-UBI (29-41) for preparation of ⁶⁸Ga labeled NOTAUBI(29-41) at hospital radiopharmacy for infection imaging. Initial clinical evaluation reveal it to be a prospective agent for imaging infection.