Insufficient supply of manufacture’s buffers/diluents in relation to the number of strips per kit has been found to have negative impact on patients’ results. Some laboratories personnel tend to use diluents from other rapid tests manufacturers such as Bioline, Unigold as well as malaria rapid diagnostic test (MRDT). This study aimed at evaluating the use of 0.01M phosphate buffered saline (PBS) as detection buffer for Alere Determine® HIV rapid test. This study was carried out at Bugando School of Medicine in Mwanza, Tanzania. A total of 300 whole blood specimens; 150 HIV positive specimens from patients attending Care and Treatment Centreand 150 HIV negative specimens were retested for HIV status using Alere Determine® HIV rapid test employing normal Alere buffer and 0.01M PBS as buffer.Of the total specimens tested; 150 (100%) of HIV positive were positive by using both Alere buffer and 0.01M PBS while 150(100%) of HIV negative samples were negative by both Alere Determine® and 0.01M PBS. The agreement between 0.01M PBS and Alere Determine® buffer was 100%. The value of kappa indicates perfect agreement between 0.01M PBS and Alere Determine® buffer (100%). A 0.01M PBS is recommended as alternative detection buffer for Alere Determine® in cases of insufficient supply. Further investigation to evaluate the suitable buffer for other rapid tests for HIV and other diseases is recommended especially in resource limited settings.