Collection of bone graft with the Reamer-Irrigator-Aspirator (RIA) system has become common practice across the field of orthopaedic surgery. While RIA bone graft is typically obtained from native long bones, grafting material can likewise be harvested from long bones that have previously undergone the placement and removal of an intramedullary nail, a process termed re-reamed RIA (RRR). The purpose of this study was to evaluate the total protein and growth factor concentrations present in native-RIA (NR) compared with RRR samples. NR and RRR bone grafts were collected intraoperatively with the RIA system and processed to evaluate both the aqueous and the hard tissue components. Total protein concentration and specific growth factors were analyzed using standard bicinchoninic acid and multiplex assays, respectively. Analyte levels were then normalized to the total amount of protein detected. Total protein levels were comparable between NR and RRR samples for both the aqueous filtrate and the hard tissue samples. When normalized, while levels of bone morphogenic protein-2 and vascular endothelial growth factor were comparable in the hard tissue component, the aqueous filtrate from the RRR sample was found to have elevated levels of growth factors, with bone morphogenic protein-2 reaching statistical significance. This study demonstrates that ample protein is found within both NR and RRR samples, with comparable or elevated levels of osteogenic growth factors found within RRR samples. Future, larger, prospective studies will be required to evaluate the osteogenic potential and clinical efficacy of NR and RRR cancellous bone grafts to validate their equivalency.