Microglia, the immune cells of the central nervous system, are dynamic and heterogenous cells. While single cell RNA sequencing has become the conventional methodology for evaluating microglial state, transcriptomics do not provide insight into functional changes, identifying a critical gap in the field. Here, we propose a novel organelle phenotyping approach in which we treat live human induced pluripotent stem cell-derived microglia (iMGL) with organelle dyes staining mitochondria, lipids, lysosomes and acquire data by live-cell spectral microscopy. Dimensionality reduction techniques and unbiased cluster identification allow for recognition of microglial subpopulations with single-cell resolution based on organelle function. We validated this methodology using lipopolysaccharide and IL-10 treatment to polarize iMGL to an "inflammatory" and "anti-inflammatory" state, respectively, and then applied it to identify a novel regulator of iMGL function, complement protein C1q. While C1q is traditionally known as the initiator of the complement cascade, here we use organelle phenotyping to identify a role for C1q in regulating iMGL polarization via fatty acid storage and mitochondria membrane potential. Follow up evaluation of microglia using traditional read outs of activation state confirm that C1q drives an increase in microglia pro-inflammatory gene production and migration, while suppressing microglial proliferation. These data together validate the use of a novel organelle phenotyping approach and enable better mechanistic investigation of molecular regulators of microglial state.