The present study was undertaken to isolate 5-hydroxytryptamine (5-HT) from crude Indian red scorpion (Mesobuthus tamulus; MBT) venom as it has not been reported yet. Therefore, with the help of bioassay (using gastric fundus contractions) and fluorimetric assay methods, the 5-HT content of crude MBT venom (CV) was determined. The concentration of 5-HT in CV by either of the methods was different (117 times). Further, a 5-HT antagonist methysergide partially blocked the CV-induced gastric fundus contractions. Hence, for isolation and determination of 5-HT in crude MBT venom, CV was subjected to Sephadex gel (G 75-125) filtration chromatography. The eluates were screened for optical density (OD) at 280 nm, lethality and gastric fundus contractions. Two peaks of enhanced gastric fundus contractions were observed between 26-70 ml and 90 -110 ml of eluate volumes. As the first peak (between 26-70 ml) eluates exhibited high OD and lethality, further analysis was not undertaken. The second peak (between 90-110 ml) eluates exhibited very low OD and were non-lethal. On bioassay, the 5-HT content of pooled eluates of second peak was 0.0398 ± 0.008 μg/ml and by fluorimetric assay it was 0.0396 ± 0.007 μg/ml. The 5-HT concentration of crude MBT venom per unit mass was 0.0175 ± 0.0035 μg/mg by bioassay and 0.0174 ± 0.0031 μg/mg by fluorimetric assay. Further, the gastric fundus contractions elicited by second peak eluates were blocked by methysergide (1.0 μM). Thus, the second peak eluates of crude MBT venom contain 5-HT and its concentration was about 0.0174 μ/mg.