Protein kinases, assembled into phosphorylation relays, transmit and process biochemical information through intracellular space for cells to respond appropriately to their environment. It remains unclear how diverse responses are encoded within a limited number of kinases involved in signal transduction. Here, we investigated the influence of pH, a fundamental property in nature, on c-Jun N-terminal kinases (JNK) which are members of the Mitogen-Activated Protein Kinase (MAPK) family. JNK signals are integral to cell responses to environmental stimuli but have complex cellular functions that remain poorly delineated. Quantitative imaging of biosensors for pH and JNK activity in live cells revealed that intracellular pH (pHi) was a significant regulator of JNK signalling. Specific manipulation of pHi within a narrow physiological range was sufficient to modify JNK activity in response to diverse stress stimuli. To determine how JNK signals ‘sense’ subtle variations in pHi we generated CRY2 fusions and utilized optogenetic seeding of protein condensates to show that pHi influenced the liquid-liquid phase separation of kinases with the JNK pathway. Specifically, we found that ASK1 and JNK2 underwent phase transitions to assemble condensates in the cytosol and this was enhanced under alkaline pHi. Interestingly, ASK1 and JNK2 condensate formation had opposing effects on their enzymatic activities (phosphorylation). In silico modelling of the differential effects of pHi-dependent condensate formation on ASK1 and JNK2 activity was sufficient to predict the signal output from the JNK pathway and revealed features of a pHi-regulated signal network that helps us predict cellular responses to cell stress. This provides improved understanding of how cellular context, such as pHi, could delineate the output and function of protein kinase pathways in response to different stimuli.