Managing contaminated waste in blood banks is a crucial process that must end with the safe disposal of blood products. In this sense, practical methods and indicators must be used to guarantee the operability of autoclave equipment and the sterility of treated waste to mitigate the risk of biological exposure and hospital biosafety. We designed a cross-sectional study to evaluate the performance the autoclaving process with Bowie & Dick test and biological indicator at the Hemotherapy and Blood Bank Unit of the Cayetano Heredia Hospital in Peru. Fifty autoclaving processes were carried out independently for the Bowie & Dick tests and biological indicators based on Geobacillus stearothermophilus spores. Autoclaving was programmed at 134°C for 3.5 minutes for the Bowie & Dick test, while, for the biological indicator, it was programmed at 121°C for 5 minutes. Both in the presence of contaminated waste. The autoclaving process evaluated by the Bowie & Dick test had a compliance rate of 80% (CI95: 66.3%–90.0%), while, by the biological indicator, 90% (CI95: 78.2%–96.7%). We did not find significant differences between the results of both tests ( P = .689; Fisher exact test). The compliance rate in the autoclaving process within the blood bank of a Peruvian Hospital is acceptable; however, there are actions for continuous improvement, especially at the vacuum level in the autoclaving process.