Retinal vein cannulation is a technically demanding surgical procedure and its feasibility may rely on using advanced surgical robots equipped with force-sensing microneedles. Reliable detection of the moment of venous puncture is important, to either alert or prevent the clinician from double puncturing the vessel and damaging the retinal surface beneath. This paper reports the first in-vivo retinal vein cannulation trial on rabbit eyes, using sensorized metal needles, and investigates puncture detection. We utilized total of four indices including two previously demonstrated ones and two new indices, based on the velocity and force of the needle tip and the correlation between the needle-tissue and tool-sclera interaction forces. We also studied the effect of detection timespan on the performance of detecting actual punctures. The new indices, when used in conjunction with the previous algorithm, improved the detection rate form 75% to 92%, but slightly increased the number of false detections from 37 to 43. Increasing the detection window improved the detection performance, at the cost of adding to the delay. The current algorithm can supplement the surgeons' visual feedback and surgical judgment. To achieve automatic puncture detection, more measurements and further analysis are required. Subsequent in-vivo studies in other animals, such as pigs with their more human like eye anatomy, are required, before clinical trials. The study provides promising results and the criteria developed may serve as guidelines for further investigation into puncture detection in in-vivo retinal vein cannulation.