Salivary alpha-amylase (sAA) enzyme functions as a digestive enzyme in many species that consume starch in their diet. Human studies have also revealed that sAA enzyme activity levels are positively correlated with the release of the stress hormone norepinephrine, allowing sAA to act as a biomarker for sympathetic nervous system activity. Recent non-human primate studies have incorporated sAA as a physiological stress marker. However, no published reports have investigated the time course of sAA from a stressful event to return to baseline levels in non-human primates. Furthermore, no validation of sAA as a stress biomarker has been reported for Japanese macaques (Macaca fuscata). This study had two primary aims: (1) to develop a systematic method for non-invasive saliva collection and, (2) to investigate sAA as a biomarker of acute stress in M. fuscata in order to better understand its acute stress-related characteristics. We developed a non-invasive method for cooperative saliva collection using positive reinforcement training (PRT) and tracked individual progress over 595 trials in ten individually housed Japanese macaques. We detected sAA enzyme in M. fuscata via kinetic reaction assay, then performed 22 acute stress tests. Four tests met conditions for interpreting sAA in response to an acute stressor and these results show that on average sAA activity rapidly increased post-stressor (mean ± SD = 4.2 ± 0.9 min) and returned to baseline shortly thereafter (10.4 ± 0.6 min). Our report reveals for the first time the temporal dynamics of sAA when applying acute stress to Japanese macaques and could be a useful tool for assessing animal welfare.