Background and purpose <p>Multifocal Electroretinogram (mfERG) applies a fast flicker-based stimulation on the human retina and is considered a valuable tool in studying the cone photoreceptor functional pathways. At the same time, the global flash paradigm of the mfERG (MOFO mfERG) is found to be advantageous in the simultaneous evaluation of retinal responses arising from outer (direct component, DC) and inner (induced component, IC) retinal levels. Incorporating a silent substitution stimulus to the MOFO mfERG remains unexplored yet has broad potential utility. The present study aimed to develop an L- and M-cone directed global flash mfERG.</p> Methods <p>A silent substitution stimulus was created appropriate for the commercially available LED monitor. Using the conventional 96% contrast MOFO mfERG as a reference, L- and M-cone directed MOFO mfERG with 19-hexagon stimulation was created and tested in 36 Chinese adults with normal colour vision. Experimental validation was conducted using high-intensity red and green light adaptation to simulate colour vision deficiency.</p> Results <p>Mathematical validation was performed by calculating and ensuring that proper cone quantal catches are achieved at both targeted and non-targeted cone photoreceptors. The cone response amplitude from participants with simulated protanopia and deuteranopia showed a reduction of up to 50% (p &lt; 0.05) following pigment bleach due to light adaptation. The mean L/M cone amplitude ratio for the Chinese adults concerning the DC and IC was 0.84 ± 0.29 and 0.77 ± 0.32, respectively, for all rings combined. The M-cone amplitudes were higher than that of the L-cone. The M-cone responses were phase-advanced or faster compared to the L-cone responses (p &lt; 0.001).</p> Conclusion <p>The L- and M-cone-directed global flash mfERG protocols may provide valuable details on the specific cone-related outer and inner retinal responses and hold extensive utility in cone-related diseases and the evaluation of colour vision.</p>