This study developed a new method for determining the sex of Periophthalmus gracilis (Eggert, 1935) fish using morphological measurements. This alternative method could potentially overcome the limitations of traditional methods, such as dissection, which is time-consuming, invasive, and can only be performed on dead or mature fish. The researchers collected 1,288 fish samples over 12 months from different coastal regions in the Vietnamese Mekong Delta. The fish were sexed based on their gonads and measured for various morphological aspects, including total length, standard length, body height, eyeglass distance, eye diameter, mouth width, and head length. The researchers found that the relationship between total length and eye distance was the most effective in distinguishing between male and female fish. Males significantly increased eye distance than females, making it a vital measurement for sex determination. The combination of eye distance, body height, and mouth width could also be used to determine the sample collection time, whether during the wet or dry season. The study highlights the importance of using non-invasive and suitable ways to determine fish sex. The alternative method can potentially help reduce the number of fish used in traditional ways, ultimately aiding in conserving fish populations. Additionally, this study provides essential information for fishery management, as it can help estimate population sizes and sex ratios, leading to more effective and sustainable management strategies.