Background and objectives: Renal biopsy is the gold standard for diagnosis of transplant rejection. However, biopsy is an invasive procedure that may adversely affect the graft. Identification of differentially expressed genes in urine by RNA Sequencing (RNA-Seq) has been proposed as a noninvasive alternative approach. Nevertheless, RNA extraction from urinary sediment is challenging due to the high rate of RNA degradation. This work aimed to optimize a protocol for urinary RNA extraction (Monteiro et al., 2016), include the DV200 index as an integrity metric and subsequently use them in different biological matrixes to obtain RNA suitable for RNA-Seq. Materials and Methods: An RNA extraction protocol involving TriReagent, glycogen, and sodium acetate, was used to extract RNA from urine, peripheral blood, and renal biopsy samples from kidney transplant patients. RNA concentration and integrity were measured. cDNA libraries were generated, sequenced, and then evaluated by primary analysis to assess the functionality of the isolated RNA. Results: RNA concentration and Integrity were suitable for RNA-Seq. In addition, sequenced libraries were clustered according to the sample type. Conclusions: The optimized protocol for RNA extraction comprising the DV200 index helped isolate urinary RNA with the quality required for next-generation sequencing. This could represent an advantageous alternative for the diagnosis and prognosis of renal allograft rejection in transplant patients.