This protocol details a method to quantify the distribution of protein and colocalization in neuronal cultures. To that end, this protocol includes itemized steps and considerations for performing immunocytochemistry, acquiring fluorescence images and quantifying multichannel fluorescence images. Success in quantifying immunostained neurons relies on the accessibility of the proteins of interest, the sensitivity and specificity of antibodies, the signal-to-noise ratio of the collected image and the sensitivity of the quantification method. In contrast to other commonly employed methods for quantification, the protocol detailed here requires manual selection of punctae and subtraction of background selected for each neurite. This approach reliably and uniquely allows for detection of proteins in low signal-to-noise ratio images, which are characteristic of developing neurons. Thus, this method serves an important niche in image analysis poorly addressed by alternative published methods. In general, immunocytochemistry requires 3.5-7 h, and one triple-immunostained neuron can be quantified in 1.5 h.