This study presents the isolation of exosomes and RNA from 20 samples of white-tailed deer serum using a commercially available kit, incorporating optimizations for RNA yield through two elutions and replicate pooling. The RNA yield from each method is reported, with six samples processed using three techniques. Seven samples underwent RNA sequencing, where total RNA was prepared for miRNA analysis using the NEBNext library preparation protocol. Adaptors were ligated to the sRNA ends, and cDNA libraries were generated through PCR enrichment. Sequencing was performed on the Illumina NovaSeq 6000 platform with 50 bp single-end reads. Data analysis involved filtering raw reads, mapping sequences against the white-tailed deer reference genome, and aligning mapped sRNA to miRBase22 for known miRNA structures. Novel miRNA predictions were made using miREvo and mirdeep2. Six samples yielded at least 1 million reads, with a Pearson’s correlation coefficient (⍴) greater than 0.7, while one sample was excluded due to insufficient sequencing depth.