Aims: To develop a simple and easy-to-use assay for detection of truely positive transformants from a yeast three-hybrid assay. Methods and Results: The yeast three-hybrid system is a new powerful system for studying RNA-protein interactions in vivo. There are, however, many reports from investigators about the difficulty in distinguishing a positive from a negative result due to the hard-to-detect differences between truely positive transformants and the negative ones. A liquid synchronous-growth culture approach has been described for all positive and negative transformants and their growth densities have been compared to each other at fixed intervals of incubation times. We have designed a simple yet effective procedure to assay for positive and negative RNA-protein interactions based on liquid culture analysis of synchronously growing yeast cells. Results obtained from this new procedure clearly show differences in positive and negative transformants after a 24-h incubation of synchronously growing transformants in liquid culture. Conclusions: The procedure mentioned in this report shows clearly the differences between positive and negative results from a three-hybrid system. Significance and Impact of the Study: The method proposed here is a clear advantage over existing methods based on measuring growth on restrictive growth medium plates by the naked eye. This method will have substantial usage with investigators using the yeast three-hybrid studies for RNA-protein interactions.