In the last few years, several attempts were made for the setting up of specific methodologies aiming to evaluate steroid contents directly in nervous tissues. The main objective of these attempts was to determine accurately changes intervening in endogenous neuroactive steroid levels during pathological situations. The present paper summarizes the reviews and original articles included in the special issue of Neurochemistry International dedicated to this important question. The issue offers a promising background for novel neuroactive steroid-based therapeutic strategies against neurodegenerative diseases.