The aim was to evaluate the potential of MitoBADY as a Raman sensor in monitoring the drug-induced differentiation of promyelocytic cells into neutrophils. Neutrophils are important immune system components, and their metabolic disorders lead to serious diseases. Primary neutrophils found in the bloodstream are short-lived cells, terminally differentiated, and unable to proliferate. Thus, the characteristics of induced differentiation of promyelocytic cells are valuable for better chemotherapy design, as it is difficult to conduct such studies in vivo or ex vivo in bone marrow environments. Alternatively, a stable in vitro model can be used to analyze the differentiation process, e.g., the HL-60 promyelocytic leukemia cell line. The standard method to confirm the promyelocytic differentiation is immunophenotyping, which is complex and laborious. Here, we propose a simple spectroscopic-based alternative that uses the Raman signal of the MitoBADY sensor to identify neutrophil-like cells. We found that the ratio of the intensity of three bands, i.e., A= (I750+I2220)/I750, B=(I2850+I2220)/I2850, and A/B, of the Raman spectra of cells incubated with the MitoBADY is a specific marker indicating the induction of neutrophil differentiation of HL-60 cells. Flow cytometry, measuring the level of the expression of the CD11b surface protein, was used as a reference method. Raman measurements allowed the classification of neutrophil-like cells with a sensitivity of 79.8 % and a specificity of 79.5 %. These values demonstrate the great potential of the proposed methodology as a rapid and reliable technique for the evaluation of the differentiation of promyelocytic cells into neutrophils.