This article proposes a new method for studying differential operators with a discontinuous weight function. It is assumed that the potential of the operator is a piecewise smooth function on the segment of the operator definition. The conditions of «conjugation» at the point of discontinuity of the weight function are required. The spectral properties of a differential operator defined on a finite segment with separated boundary conditions are studied. The asymptotics of the fundamental system of solutions of the corresponding differential equation for large values of the spectral parameter is obtained. With the help of this asymptotics, the «conjugation» conditions of the differential operator in question are studied. The boundary conditions of the operator under study are investigated. As a result, we obtain an equation for the eigenvalues of the operator, which is an entire function. The indicator diagram of the eigenvalue equation, which is a regular polygon, is studied. In various sectors of the indicator diagram, the asymptotics of the eigenvalues of the investigated differential operator is found. The formula for the first regularized trace of this operator by using the found asymptotics of the eigenvalues by the Lidsky–Sadovnichy method is obtained. In the case of the passage to the limit, the resulting formula leads to the trace formula for the classical operator with a smooth potential and constant weight function.