Background: Quantifying the relative abundance of different neurotransmitters in the myenteric plexus has proved challenging using conventional immunocytochemical approaches. Here, we present a new method of quantifying neurotransmitter content of an important enteric signalling molecule, serotonin (5-HT), in the myenteric plexus of guinea pig colon under different experimental conditions. Methods: Sections of guinea pig distal colon were exposed to different conditions including changes in temperature, dissection protocol, stimulation with faecal pellet distension and exogenous 5-HT. Sections were fixed and immuno-labelled for 5-HT. 5-HT staining density was quantified within myenteric plexus ganglia using defined settings and an analysis approach that uses threshold settings allowing for variances in background and tissue staining intensities and which calculates the area of tissue containing 5-HT above these thresholds. Key Results: No differences were found in 5-HT immunoreactivity in the myenteric plexus when compared between tissues that were freshly fixed, undissected, or with mucosa and submucous plexus dissected away at either 4 or 37 °C. Increased myenteric plexus 5-HT density was observed in preparations repeatedly stimulated using faecal pellet stimulation prior to fixation. Furthermore, exogenous 5-HT also increased 5-HT density. Conclusions & Inferences: We demonstrate that quantitative differences in 5-HT immunoreactivity can be characterized using immunohistochemistry. This approach may be applied to measuring other neurotransmitter(s) within the enteric nervous system. While 5-HT is present in the guinea-pig enteric ganglia, this is not due to accumulation via in vitro handling and release from the mucosa, and furthermore, repeated colonic stimulation via distension increases 5-HT in the myenteric plexus.