Histamine is an autacoid that exerts multiple functions in physiologic and pathophysiological processes. Histamine analysis in biological samples or certain high risk food is of a great importance for disease diagnosis or monitoring the quality of food products. The aim of this study was to evaluate the effect of sample condition on histamine detecting capability of reversed-phase ion-pairing HPLC, a reliable and less difficult method comparing some other HPLC techniques. Histamine detectability was evaluated in a buffer system relevant to the food and bio-samples; under pH 5.0, 7.0 and 9.0 while regulating temperature at 25°C, 37°C and 100°C. The results showed that acidifying the sample, perhaps due to development of more positive charged histamine, provides a better resolution either at 25°C or 37°C (P<0.05). The study suggested that maximum ionization of histamine fulfils the requirements of ion-pair RF-HPLC to act as a potent and selective technique in histamine separation and quantification.