The separation and detection of isomers remains a challenge for many areas of mass spectrometry. This article highlights laser photodissociation and ion mobility strategies that have been recently deployed to meet this challenge with focus on small molecule isomers including protonation isomers, structural isomers, conformation isomers and new studies emerging on chiral isomers. Laser techniques span UV and visible laser photodissociation, time-resolved pump–probe schemes and application of laser hole-burning arrangements to assign isomers within selected ion populations. Also surveyed are applications of ion mobility strategies to separate isomers followed by laser spectroscopic techniques to assign the separated ions. Ultimately, with ongoing refinement in hardware and methods, there are clear pathways forward for laser and mass spectrometry techniques to make decisive breakthroughs in understanding how isomeric details affect biological processes, physiology and disease.