Honey and other bee products may contain residues of different substances, including pesticides, which is considered a public health problem. In addition, they characterize risks to the health of Apis mellifera, which have been showing an increasing decline in their populations. There are many protocols for identifying pesticides in bee products which, in general, are complex matrices whose results of routine investigations in control laboratories are rarely disclosed. In this sense, the objective of the present study was to determine the presence of residues of glyphosate and its metabolite aminomethylphosphonic acid (AMPA) in honey, as well as its effect on the strength of the hive of A. mellifera. Samples were collected from hives experimentally exposed to food containing a sublethal dose of Roundup® and conducted by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry (HILIC-MS/MS). The AMPA content was lower than the method’s detection limit in honey samples from hives that received food containing the herbicide. It was possible to quantify glyphosate one week after the last artificial feeding (R1 8.45 ± 1.09 µg g−1; R2 8.15 ± 2.14 µg g−1; R3 23.90 ± 2.95 µg g−1). In a hive sample fed for more than four weeks, glyphosate was present in lower concentrations (3.12 ± 0.89 µg g−1) with no detection of AMPA. From the analysis of the strength of the hives, we observed a decrease in the population of adult individuals and the brood area, the absence of a queen, and no construction of royal cells by the workers in the hives of the Roundup® treatment in comparison to the control group, in which the hives remained with queen size, high adult and brood population, and food stock. Although present, glyphosate did not undergo degradation in honey during the evaluated period. Thus, we could infer that the presence of Roundup® in bee feed may be present in honey, representing a risk to consumers’ health and economic damage to beekeepers. This is the first study that evaluated the effect of glyphosate residues on hive strength, contributing to the understanding of the Roundup® mode of action in different aspects that affect the survival of colonies under field conditions.