Ex vivo porcine lung immersed in e‐liquid was investigated in‐depth using confocal Raman micro‐spectroscopy to assess the e‐liquid influence on the lung. It was found that lung‐related Raman band intensities at 1002, 1548, 1618 and 1655 cm−1 increased after first and second treatments except the surface, which was attributed to the well‐known optical clearing effect due to alveoli filling with e‐liquid resulting in light scattering reduction. The autofluorescence enhancement was explained by oxidative stress induced in lung during exposure to e‐liquid. Moreover, e‐liquid induced collagen dehydration was revealed by the I937/I926 Raman band intensity ratio change. The effect was enhanced after the second treatment of the same lung tissue that indicates the possibility of multi‐step OC treatment. We hypothesize that the nicotine‐flavour free e‐liquids containing glycerol and propylene glycol could potentially be used in clinical protocols as optical clearing agent for enhanced in‐depth Raman‐guided bronchoscopy.This article is protected by copyright. All rights reserved.