The retention characteristics of five stationary phases were tested by using a selection of 5′-mononucleotides and nucleosides with the aim to develop a simple, rapid and sensitive reversed-phase liquid chromatography method without ion-pair reagent usage. The method was optimized by changes in temperature, pH and ionic strength on a column showing a superior performance. The mobile phase consisted of a mixture of 0.05 M phosphate buffer and methanol, delivered at a flow rate of 0.4 mL/min and based on a gradient program. UV detection was used at a 254 nm wavelength. The method was validated for a quantitative analysis of 5′-mononucleotides and nucleosides in wild edible mushrooms. For all nucleosides and nucleotides, the LOD and LOQ were less than 0.02 and 0.07 μg/mL, respectively. Validation parameters yielded recovery rates between 68.6 and 98.2%, with a precision expressed as a relative standard deviation of 7.6–15.3%. The content of 5′-mononucleotides and nucleosides was determined for 10 samples of wild edible mushrooms found in Croatia and, accordingly, the equivalent umami concentrations were calculated.