The motility of macrophages in response to microenvironment stimuli is a hallmark of innate immunity, where macrophages play pro-inflammatory or pro-reparatory roles depending on their activation status during wound healing. Cell size and shape have been informative in defining macrophage subtypes. Studies show pro- and anti-inflammatory macrophages exhibit distinct migratory behaviors, in vitro, in 3D, and *in vivo, *but this link has not been rigorously studied. We apply both morphology and motility-based image processing approaches to analyze live cell images consisting of macrophage phenotypes. Macrophage subtypes are differentiated from primary murine bone marrow-derived macrophages using a potent lipopolysaccharide (LPS) or cytokine interleukin-4 (IL-4). We show that morphology is tightly linked to motility, which leads to our hypothesis that motility analysis could be used alone or in conjunction with morphological features for improved prediction of macrophage subtypes. We train a support vector machine (SVM) classifier to predict macrophage subtypes based on morphology alone, motility alone, and both morphology and motility combined. We show that motility has comparable predictive capabilities to morphology. However, using both measures can enhance predictive capabilities. While motility and morphological features can be individually ambiguous identifiers, together they provide significantly improved prediction accuracies (>79%) from a training dataset of ~250 cells tracked over time using only phase contrast time-lapse microscopy. Thus, the approach combining cell motility and cell morphology information can accurately assess functionally diverse macrophage phenotypes quickly and efficiently. Our approach offers a cost-efficient and high-throughput method for screening biochemicals targeting macrophage polarization.

• Macrophage Differentiation: Naïve BMDMs (M0) were differentiated into M1 or M2 subtypes. For M1 differentiation, 100 ng/ml lipopolysaccharide (LPS) was used. For M2 differentiation, 20 ng/ml recombinant mouse interleukin-4 (IL-4) was employed.

• Culturing and Activation: The macrophages were seeded in 24-well tissue culture-treated plates and activated as detailed in the accompanying manuscript.

• Imaging: Time-lapse contrast images were captured 48 hours post-stimulation using a Carl Zeiss Observer Z1 inverted microscope with a motorized stage and incubation chamber. Imaging was done using the MetaMorph program with a Retiga R6 CCD camera, under 10× or 20× objective lenses.