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Deep wound infection after spinal surgery is a potentially devastating complication and is associated with higher morbidity, mortality and healthcare costs. Different measures including intraoperative application of vancomycin powder to wounds have been employed previously to decrease the infection rate. The primary objective of this ongoing clinical study is to evaluate the systemic uptake of prophylactically applied vancomycin in instrumented spinal fusion surgery. Secondary outcomes are to show any side effects including nephrotoxicity related to its local application and record superficial and deep wound infections. A prospective study has been designed to recruit consecutive patients, between September 2013 and September 2014, operated by a single surgeon. All patients undergoing instrumented spinal fusion surgery (elective and trauma) in a single institution over a 12-month period were included. One gram of vancomycin powder was applied to the subfascial layer, and serum levels were measured at 6, 12 and 24 h post-administration. All patients routinely had renal functions checked postoperatively to evaluate nephrotoxic effects. A second cohort of patients was then recruited to apply 2 g of vancomycin subfascially. The patients were followed up for a 2-year period. Twenty-four patients, both trauma and elective, had 1 g of vancomycin powder applied to the subfascial layer. Twenty-eight patients had 2 g of vancomycin powder applied to the subfascial layer. Four patients reached systemic levels in the 2-g group; however, only one patient had clinically detectable but nonsignificant levels in the 1-g group. There were no adverse effects detected. This study demonstrates that systemic uptake of vancomycin after local application to the wound is negligible for the vast majority of patients. However, it has shown clinical and biochemical safety for its use and remains a cost-effective and low-risk strategy to combat surgical site and deep wound infections.