We studied the validation for drug content of a new insulin cartridge (Humacart 3/7, 3.0 ml cartridge®). The concentrations of total insulin, Zn, m-cresol, and phenol in the Humacart 3/7, 3.0 ml cartridge® were consistent with the levels described in the interview form. To study the relationship between the mixing (0, 5, and 10 times) and the uniformity of the insulin suspension, a Humacart 3/7, 3.0 ml cartridge® was set into the insulin delivery device specifically designed for this cartridge (Autopen®). The total insulin concentrations of this preparation through Autopen® varied from 30 to 150 U/ml without mixing, so mixing more than 10 times was necessary to make the suspension uniform. The total insulin concentrations through Autopen® decreased with increased standing times after thorough mixing; thus the Humacart 3/7, 3.0 ml cartridge® must be injected as soon as possible after mixing. The dimeric-hexameric insulin concentration in the Humacart 3/7, 3.0 ml cartridge® was 10 times higher than the monomeric insulin concentration. Furthermore, the insulin concentration through Autopen® with a 31-gauge needle was the same as the insulin concentration using a 30-gauge needle. These results indicate that the characteristics of a Humacart 3/7, 3.0 ml cartridge® are equivalent to that of a Humacart 3/7, 1.5 ml cartridge®.