Single-shot, two-frame, π-shifted spatially multiplexed interference microscopy (π-SMIM) is presented as an improvement to previous SMIM implementations, introducing a versatile, robust, fast, and accurate method for cumbersome, noisy, and low-contrast phase object analysis. The proposed π-SMIM equips a commercially available nonholographic microscope with a high-speed (video frame rate) enhanced quantitative phase imaging (QPI) capability by properly placing a beam-splitter in the microscope embodiment to simultaneously (in a single shot) record two holograms mutually phase shifted by π radians at the expense of reducing the field of view. Upon subsequent subtractive superimposition of holograms, a π-hologram is generated with reduced background and improved modulation of interference fringes. These features determine superior phase retrieval quality, obtained by employing the Hilbert spiral transform on the π-hologram, as compared with a single low-quality (low signal-to-noise ratio) hologram analysis. In addition, π-SMIM enables accurate in-vivo analysis of high dynamic range phase objects, otherwise measurable only in static regime using time-consuming phase-shifting. The technique has been validated utilizing a 20  ×    /  0.46 NA objective in a regular Olympus BX-60 upright microscope for QPI of different lines of prostate cancer cells and flowing microbeads.