This study was designed to evaluate the maximal amount of blood that can be safely collected in healthy, adult male and female cynomolgus macaques for 4 consecutive weeks with minimal effect on animal wellbeing. General guidelines for blood collection volumes in laboratory animals are not species-specific, and currently there are few evaluations of blood collection in macaques. In this study, blood was removed at 7.5%, 10%, 12.5%, 15%, or 17.5% of total blood volume (TBV) for 4 consecutive weeks. Hematologic parameters and body weights were evaluated immediately prior to each blood collection time point and for an additional 4 consecutive weeks following the last collection. Male and female macaques tolerated removal of as much as 15% TBV with minor clinical effects, whereas macaques in the 17.5% TBV group exhibited an increased incidence of emesis and anorexia during the first 24 h after blood collection. According to these results, we recommend collecting no more than 15% TBV weekly for 4 consecutive weeks from healthy, adult male and female cynomolgus macaques.