Green auto-fluorescence (GAF) of different age groups of mouse blood erythrocytes was determined by using a double in vivo biotinylation (DIB) technique that enables delineation of circulating erythrocytes of different age groups. A significant increase in GAF was seen for erythrocytes of old age group (age in circulation > 40 days) as compared to young erythrocytes (age < 15 days). Erythrocytes are removed from blood circulation by macrophages in the reticulo-endothelial system and depletion of macrophages results in an increased proportion of aged erythrocytes in the blood. When mice were depleted of macrophages for 7 days by administration of clodronate loaded liposomes, the overall GAF of erythrocytes increased significantly and this increase could be ascribed to an increase in GAF of the oldest population of erythrocytes. Using the DIB technique, the GAF of a cohort of blood erythrocyte generated during a 5 day window was tracked in vivo. GAF of the defined cohort of erythrocytes remained low till 40 days of age in circulation and then increased steeply till the end of the life span of erythrocytes. Taken together our results provide evidence for an age dependent increase in the GAF of blood erythrocytes that is accentuated by depletion of macrophages. Kinetics of changes in GAF of circulating erythrocytes with age has also been defined.