To compare and evaluate two methodologies, entire-sampling and micro-sampling for the harvesting of vitreous humor, the vitreous humor of rabbits were sampled with the two methods respectively, and the concentrations of calcium, chlorine, potassium, sodium and phosphorus of the were measured. The results showed that the differences in the variance coefficient and two-eye concentrations of micro-sampled specimens were less than those of the entire-sampled specimens. In the micro-sampling group, the concentrations of repeated micro-sampling showed no differences among different groups (P＞0.05) and the intra-ocular fluid dynamics did not have significant influence on post-mortem sampling. The sampling technique may affect the concentrations of specimen collected.Our study suggests that micro-sampling is less influenced by the human factor and is reliable, reproducible, and more suitable for forensic investigation.