During metamorphosis in Drosophila, muscles undergo developmentally regulated remodeling, which involves cell death of obsolete and atrophy of persistent muscles. Thanks to the ability to perform live imaging of muscle development in transparent pupae and the power of genetics, metamorphosis in Drosophila can be used as a model to study the regulation of skeletal muscle mass. We performed targeted gene perturbation in muscles and acquired 3D time series images of muscle development in metamorphosis using laser scanning confocal microscopy. To help us quantify the phenotypic effects of gene perturbations in large number of images, we designed an ImageJ based Fly Muscle Analysis tool (FMAj) and MySQL frameworks for image processing and data storage, respectively. The image analysis pipeline of FMAj is divided into three modules. The first module assist the user in adding annotations to the time-lapse datasets, such as genotype, experimental parameters and temporal reference points, which are used to compare different datasets. The second module performs segmentation and feature extraction of muscles cell and nuclei. The third module performs comparative quantitative analysis of muscle phenotypes.  We demonstrated our tool in the time series phenotypic characterization of two atrophy related genes, which were silenced by RNA interference. Reduction of the Drosophila TOR homolog resulted in atrophy, while inhibition of the autophagy factor Atg9 led to hypertrophy and abnormal morphology of muscle fibers. By applying statistical analysis we could show statistically significant differences in muscle diameter between controls and the two types of gene perturbation. Our in vivo imaging experiments revealed that genes involved in TOR signalling and autophagy are not only conserved in sequence between mammals and Drosophila, but also perform similar function in regulating muscle mass. Extending our approach to a genome-wide scale has the potential to identify new genes involved in muscle size regulation.