Cryo-EM is well placed to study protein interactions and dynamics with the ability to separate and determine the structure of different conformational states in silico from a single data set. In a typical cryo-EM experiment, the sample is at equilibrium prior to vitrification and the range of conformational states is limited to those accessible by thermal energy. In contrast, time-resolved cryo-EM (trEM) aims to fixate the sample prior to equilibrium so that desired conformations that are present after a defined period of time can be isolated. For the majority of biochemical systems, reactions (or the return to an equilibrium or steady state) are on the millisecond timescale, making trEM difficult with conventional approaches [1]. However, with purpose-built trEM instruments, it is possible to start and quench (by vitrification) a reaction in the ms timeframe. The Henderson group successfully combined electron diffraction with photoactivation to study the photocycle of bacteriorhodopsin whereas Unwin & Berriman were among the first to demonstrate the approach of spraying substrate onto a pre-prepared grid before freezing and quenching the reaction [2, 3, 4]. Following this and especially since the ‘resolution revolution’, there have been a number of different bespoke setups designed for trEM including those by our own group and the Frank, Carragher and Enchev groups amongst others [5, 6∗, 7∗∗, 8]. Excellent overviews of the different approaches for trEM and its applications have been given elsewhere [9,10]. This is a developing area but already trEM has been used to great effect to study the conformational changes associated with the formation of the 70S ribosome elongation complex and visualising the reaction intermediates of RecA filament growth in the ms timescale [7,11] or the formation of a double hexamer complex of the MCM helicase in the seconds timescale [12]. Here we summarise some of the current approaches and focus on the limitations and strengths of each method before discussing the future perspectives and challenges that remain in the field.