Tenofovir disoproxil fumarate is a pro-drug of the active metabolite tenofovir widely used against the HIV1, HIV2, and Hepatitis B virus. Several studies have been conducted and found kidney injury associated with tenofovir exposure. High tenofovir plasma concentration correlated with kidney injury in tenofovir-exposed patients. The present study developed and validated a simple and cost-effective LC/MS/MS method to determine tenofovir level in human plasma. A small plasma volume of 80 μl is utilized for the sample preparation. The samples were separated by Luna C18 (100 mm × 2.0 mm, 3 μm) using gradient elution with a mobile phase consisting of water (containing 0.1% formic acid) and acetonitrile (90:10, v/v). The detection was achieved through multiple reaction monitoring using positive ionization mode on the triple quadrupole mass spectrometer with a run time of 10 min. The monitoring transitions were set at m/z 288.0 → 176.1 and 136.1 for tenofovir and m/z 226.1 → 152.0 for acyclovir (as the internal standard). This standard curve was linear from 10 to 640 ng/ml, with the lower limit of quantification of 10 ng/ml. The inter- and intra-day precision results were less than 12.3% and their accuracies were within the acceptable range of 84.9–113.1%. The validated method was successfully applied to the study of tenofovir induced kidney injury in HIV-1 infected patients taking 300 mg once daily for more than 4 weeks.