Purpose: To develop a method of injecting a volume up to 50% of the lacrimal gland (LG) volume while minimizing patient discomfort and maximizing accurate drug delivery. Herein we describe a series of ultrasound (US)-guided transcutaneous injections in the LG and discuss the safety and feasibility of this technique. Methods: Ultrasonography was performed in 40 patients with aqueous deficient dry eye disease using a GE Logic E10 (Milwaukee, Wisconsin, USA) US machine with a 6-24 MHz transducer. US was performed by 2 medical experts in ultrasonography. We recorded the injection and observed an enlargement of the LG ensuring delivery within the LG before the needle was removed. Assessment of injection-related adverse event was performed immediately after the injection. Results: The position of the injection needle within the LG was documented in all 40 patients. Injection of the stem cells and vehicle (N = 20) or solely vehicle (N = 20) led to an enlargement of the glandular structures in all cases. No serious adverse reactions related to the injections were observed. Conclusion: US-guided injection into the LG enables injection on a closed eye causing minimum patient discomfort and maximum certainty of accurate drug delivery. US can provide real-time images and may be used to safely guide the needle ensuring correct placement and injection within the gland capsule. This reduces the risk of injury to the eye and adjacent structures and makes a precise transcutaneous injection possible.