Within a cell, the regulation of organelle positioning is considered to be critical in spatio-temporal responses. The position of late endocytic organelles (named here lysosomes for simplicity) is tightly controlled and has a functional impact on processes like endocytosis, phagocytosis and autophagocytosis. The cytoplasmic distribution profile of lysosomes can be easily determined in cells where the cytoplasm/nuclear ratio in a cross-section area is high. However, determining lysosomal position in cells with lower cytoplasm/nuclear ratio, such as macrophages is more challenging. Here, we describe a method that can be efficiently and accurately used to determine the position of organelles in macrophages using confocal microscopy in two-dimensional (2D) images. Using this approach in macrophages, we confirmed previous observations in epithelial cells that both changes in cytoplasmic pH and the levels of active Rab34 induced a re-distribution of lysosomes to the cell centre or periphery. Noteworthy is that this Rab34-dependent re-distribution of lysosomes did not significantly affect the spatial distribution profile of phagolysosomes in the cytoplasm. We conclude that although Rab34 regulates both lysosomal positioning and lysosome to phagosome fusion, the latter effect is not due to the regulation of the cytoplasmic accessibility of lysosomes to phagosomes by Rab34.