Cell assemblies are difficult to observe because they consist of many neurons. We aimed to observe cell assemblies based on biological statistics, such as synaptic connectivity. We developed an estimation method to estimate the activity and synaptic connectivity of cell assemblies from spike trains using mathematical models of individual neurons and cell assemblies. Synaptic transmissions were averaged to generate postsynaptic currents with the same timing and waveform but different amplitudes, as the number of presynaptic neurons was large. We estimated the average synaptic transmission and synaptic connectivity from active cell assemblies based on the stochastic prediction of membrane potentials and verified the estimation ability of the average synaptic transmission and synaptic connectivity using the proposed method on simulated neural activity. Different cell assembly activities evoked by electrical stimuli were correctly sorted into various clusters in experiments using rat cortical neurons cultured on microelectrode arrays. We observed multiple cell assemblies from the spontaneous activity of rat cortical networks on microelectrode arrays, based on the synaptic connectivity patterns estimated by the proposed method. The proposed method was superior to the conventional method for detecting the activity of multiple cell assemblies. Using the proposed method, it is possible to observe multiple cell assemblies based on the biological basis of synaptic connectivity. In summary, we report a novel method to observe cell assemblies from spike train recordings based on the biological basis of synaptic connectivity, rather than merely relying on a statistical method.