Background: Environmental and physiological factors influence human ionom, which requires the development of reliable region-specific reference intervals (RIs). This study aimed to establish population-based RIs of copper (Cu), zinc (Zn), selenium (Se), rubidium (Rb), and magnesium (Mg), in relation to age, sex, and physical activity. Methods: The RIs were established according to the CLSI EP28-A3c guidelines. A reference group of 120 directly selected healthy volunteers of Bulgarian nationality (60 males and 60 females, aged 18 to 65 years) were included in the study. The participants were separated into two groups: low (2 h/week, n=60) physical activity. Serum levels of Cu, Zn, Se, Rb, and Mg (sCu, sZn, sSe, sRb, sMg), were determined via collision cell inductively coupled plasma mass spectrometry (ICP-MS). Results: The RIs in this study are generally comparable to those in other countries. However, 43% of the participants had sSe lower than the threshold of 80 µg/L, correlating with the optimal expression of key selenoproteins. A significant sex-related difference was found for Cu (male vs female, p = 0.003), as well as an age-related significant difference for Cu between the groups of women below and above 30 years of age (p = 0.001). Conclusions: In this study, we verified the RIs of sCu, sZn, sSe, and sMg for the Bulgarian population with ICP-MS. A RI of sRb, missing up to the present was established, thus filling the gap. Specific sex-, age-, and physical activity-related associations of the elements were elucidated.