Systems and methods for focusing optics are disclosed herein. In some embodiments, methods are disclosed for focusing an optical device, wherein the methods can include: collecting light from a region of an object to be imaged with an objective lens, said region having a feature with a known geometric characteristic; splitting the collected light into a first portion and a second portion, and directing said first portion through a weak cylindrical lens to a focusing sensor, and directing said second portion to an imager; observing, with said focusing sensor, a shape of the feature; focusing the optical device by moving at least one of the objective lens and the object to be imaged until the observed shape of the feature has a predetermined relationship to the known geometric characteristic. In some embodiments, the feature can be a fluorescent bead. In some embodiments, the splitting step can be accomplished with a dichroic mirror. In other embodiments, the splitting step can be accomplished with a partial mirror. In some embodiments, the known geometric characteristic of the feature can be substantially spherical, the observed shape can be an oval, and the predetermined relationship can be an allowable aspect ratio of the oval. In some embodiments, the allowable aspect ratio can be approximately one.